Cristiana Boi

Extracellular vesicles (EVs) are nanoparticles released by cells and potential drug carriers. However, their translation is limited by the absence of standardized, scalable purification methods and low yields. Drug loading creates an additional downstream challenge, namely the removal of unbound drug. Here, we demonstrate that a single tangential flow filtration process can simultaneously isolate whey-derived EVs (wEVs) and remove residual unbound drugs from the whey matrix. Bovine milk whey was used as the EV source, and curcumin (Cur) as the model drug. Passive loading, followed by constant-volume diafiltration and concentration in one ultrafiltration module, removed 98.9 ± 0.0% of the whey impurities, together with most of the residual free Cur. The process recovered 1.1 × 10¹³ particles per mL of processed whey and 10.9 ± 0.1% of the added Cur, about one third of which was associated with the vesicles. wEVs and Cur-wEVs showed comparable particle concentration, size, and ζ potential, indicating preserved vesicle integrity. At an equal total amount of Cur, Cur-wEVs exhibited higher intracellular Cur accumulation and greater inhibition of SK-N-AS neuroblastoma cell proliferation than free Cur (IC₅₀ 16.34 μM vs. 31.14 μM). Combining loading and purification in a single membrane operation addresses a major bottleneck in EV-based drug delivery.

Adeno‐associated viruses (AAVs) are the leading vectors for in vivo gene therapy, fueling a growing demand for scalable and robust biomanufacturing processes. Yet, the impact of cell lysis conditions on the performance of midstream and downstream unit operations across serotypes remains poorly characterized. This study presents a serotype‐resolved investigation of cell lysis and its cascading effects on AAV2, AAV5, AAV8, and AAV9 processing. A panel of lysis methods including inorganic agents, non‐ionic and ionic detergents, and mechanical disruption was evaluated using an analytical framework of at‐ and off‐line assays to quantify AAV yield, purity, and activity at each stage. Our results reveal pronounced serotype‐specific differences: cetrimonium bromide (CTAB) delivered the highest transduction output of any condition evaluated for AAV8 (2.25 × 10 ⁹ TU/mL) at 55.0% capture step yield, while sodium deoxycholate achieved ~ 98% host cell protein (HCP) reduction for AAV9 (HCP log reduction value 1.60) and the highest functional enrichment for AAV5 (6.73‐fold) and AAV8 (5.80‐fold). AAV2 proved uniquely challenging, with CTAB enabling the highest tangential flow filtration (TFF) yield. Acid precipitation paired with high salt addition reduced aggregation and improved permeate flux for AAV5 and AAV9. TFF performance was governed by the detergents' physicochemical properties: micelle‐forming agents generated multicomponent complexes that fouled membranes in condition‐ and serotype‐specific patterns. Rheological characterization revealed that all clarified lysates converged to the viscosity of the media after filtration, supporting transferable TFF parameters. Mixed‐mode chromatography using a peptide‐functionalized resin amplified lysis‐imprinted differences, decoupling step yield from functional enrichment. Bayesian optimization of the chromatographic step improved purification outcomes: for AAV8 residual HCPs in the eluate decreased > 300‐fold, from 0.91 mg/mL to 3 μg/mL, while for AAV9 the yield rose to ~95% at residual HCP as low as 14 μg/mL, demonstrating that purity and recovery are co‐optimizable when lysis, midstream, and downstream conditions are rationally paired.

Early detection of bacteria in food products is crucial for protecting human health and reducing economic losses. Although conventional methods, such as culture‐based techniques, nucleic acid sequence‐based detection, and immunoassays, are generally accurate, they are often time‐consuming, labor‐intensive, and require specialized equipment. In response to these limitations, sensing technologies emerge as simple, rapid, and cost‐effective alternatives. Among these technologies, graphene gains significant attention due to its unique electrical properties and biocompatibility, making it a promising material for bacterial detection. While most efforts focus on functionalizing sensors for specific targets, recent studies demonstrate that non‐functionalized graphene can effectively detect bacteria, enabling simpler and more affordable sensor design. This review provides a comprehensive analysis of non‐functionalized electrical graphene‐based bacterial sensors developed thus far. It evaluates and discusses the influence of graphene properties, bacterial activity, sensor design, and operational conditions on sensor performance. Additionally, the review emphasizes the need for further research to improve the reproducibility and scalability of reliable sensors and to develop sensitive, selective, and physiochemically stable devices.

Cellular Al₂TiO₅–Al₂O₃ composite ceramic monoliths were prepared and tested as a potential stationary phase for process chromatography. The material was characterized by measuring the pore size distribution and hydraulic permeability. The interstitial porosity, the axial dispersion coefficient, and the height equivalent to a theoretical plate were calculated by pulse tests under nonbinding conditions. The surface of the ceramic material was activated with graphene oxide and then functionalized with Cibacron Blue F3GA. The dynamic binding capacity of the functionalized columns was measured in chromatographic cycles by using bovine serum albumin (BSA) as the target molecule. The experiments showed that the proposed monoliths have a well-defined porous structure, leading to particularly interesting flow properties for chromatographic bioseparations, such as very high permeability and convection, as the main mass transport phenomenon. These results are encouraging for a possible future optimization of the functionalization procedure toward the development of efficient convective media for protein purification.

Electrospun regenerated cellulose (RC) nanofiber membranes were prepared starting from cellulose acetate (CA) with different degrees of substitution. The process was optimized to obtain continuous and uniformly sized CA fibers. After electrospinning, the CA membranes were heat-treated to increase their tensile strength before deacetylation to obtain regenerated cellulose (RC). Affinity membranes were obtained by functionalization, exploiting the hydroxyl groups on the cellulose backbone. 1,4-Butanediol-diglycidyl ether was used to introduce epoxy groups onto the membrane, which was further bioconjugated with the anti-CD63 antibody targeting the tetraspanin CD63 on the extracellular vesicle membrane surface. The highest ligand density was obtained with an anti-CD63 antibody concentration of 6.4 µg/mL when bioconjugation was performed in carbonate buffer. The resulting affinity membrane was tested for the adsorption of extracellular vesicles (EVs) from human platelet lysate, yielding a very promising binding capacity above 10 mg/mL and demonstrating the suitability of this approach.