The interactions that nanoparticles have with blood proteins are crucial for their fate in vivo. Such interactions result in the formation of the protein corona around the nanoparticles, and studying them aids in nanoparticle optimization. Quartz crystal microbalance with dissipation monitoring (QCM-D) can be used for this study. The present work proposes a QCM-D method to study the interactions on polymeric nanoparticles with three different human blood proteins (albumin, fibrinogen and γ-globulin) by monitoring the frequency shifts of sensors immobilizing the selected proteins. Bare PEGylated and surfactant-coated poly-(D,L-lactide-co-glycolide) nanoparticles are tested. The QCM-D data are validated with DLS and UV-Vis experiments in which changes in the size and optical density of nanoparticle/protein blends are monitored. We find that the bare nanoparticles have a high affinity towards fibrinogen and γ-globulin, with measured frequency shifts around −210 Hz and −50 Hz, respectively. PEGylation greatly reduces these interactions (frequency shifts around −5 Hz and −10 Hz for fibrinogen and γ-globulin, respectively), while the surfactant appears to increase them (around −240 Hz and −100 Hz and −30 Hz for albumin). The QCM-D data are confirmed by the increase in the nanoparticle size over time (up to 3300% in surfactant-coated nanoparticles), measured by DLS in protein-incubated samples, and by the trends of the optical densities, measured by UV-Vis. The results indicate that the proposed approach is valid for studying the interactions between nanoparticles and blood proteins, and the study paves the way for a more comprehensive analysis of the whole protein corona.

Krabbe disease (KD) is a rare disorder arising from the deficiency of the lysosomal enzyme galactosylceramidase (GALC), leading to the accumulation of the cytotoxic metabolite psychosine (PSY) in the nervous system. This accumulation triggers demyelination and neurodegeneration, and despite ongoing research, the underlying pathogenic mechanisms remain incompletely understood, with no cure currently available. Previous studies from our lab revealed the involvement of autophagy dysfunctions in KD pathogenesis, showcasing p62-tagged protein aggregates in the brains of KD mice and heightened p62 levels in the KD sciatic nerve. We also demonstrated that the autophagy inducer Rapamycin (RAPA) can partially reinstate the wild type (WT) phenotype in KD primary cells by decreasing the number of p62 aggregates. In this study, we tested RAPA in the Twitcher (TWI) mouse, a spontaneous KD mouse model. We administered the drug ad libitum via drinking water (15 mg/L) starting from post-natal day (PND) 21-23. We longitudinally monitored the mouse motor performance through grip strength and rotarod tests, and a set of biochemical parameters related to the KD pathogenesis (i.e. autophagy markers expression, PSY accumulation, astrogliosis and myelination). Our findings demonstrate that RAPA significantly enhances motor functions at specific treatment time points and reduces astrogliosis in TWI brain, spinal cord, and sciatic nerves. Utilizing western blot and immunohistochemistry, we observed a decrease in p62 aggregates in TWI nervous tissues, corroborating our earlier in-vitro results. Moreover, RAPA treatment partially removes PSY in the spinal cord. In conclusion, our results advocate for considering RAPA as a supportive therapy for KD. Notably, as RAPA is already available in pharmaceutical formulations for clinical use, its potential for KD treatment can be rapidly evaluated in clinical trials.

Krabbe disease is a rare neurodegenerative disease with an autosomal recessive character caused by a mutation in the GALC gene. The mutation leads to an accumulation of psychosine and a subsequent degeneration of oligodendrocytes and Schwann cells. Psychosine is the main biomarker of the disease. The Twitcher mouse is the most commonly used animal model to study Krabbe disease. Although there are many references to this model in the literature, the lipidomic study of nervous system tissues in the Twitcher model has received little attention. This study focuses on the comparison of the lipid profiles of four nervous system tissues (brain, cerebellum, spinal cord, and sciatic nerve) in the Twitcher mouse compared to the wild-type mouse. Altogether, approximately 230 molecular species belonging to 19 lipid classes were annotated and quantified. A comparison at the levels of class, molecular species, and lipid building blocks showed significant differences between the two groups, particularly in the sciatic nerve. The in-depth study of the lipid phenotype made it possible to hypothesize the genes and enzymes involved in the changes. The integration of metabolic data with genetic data may be useful from a systems biology perspective to gain a better understanding of the molecular basis of the disease.

Twitcher (Twi) is a neurological Krabbe disease (KD, or globoid cell leukodystrophy) spontaneous mutant line in mice. The genome of the Twi mouse presents a single nucleotide polymorphism (SNP), leading to an enzymatically inactive galactosylceramidase (Galc) protein that causes KD. In this context, mouse Twi genotyping is an essential step in KD research. To date, the genotyping method used is labor-intensive and often has ambiguous results. Here, we evaluated a novel protocol for the genotype determination of Galc mutation status in Twi mice based on the allele-discrimination real-time polymerase chain reaction (PCR). Here, DNA is extracted from Twi mice (n = 20, pilot study; n = 120, verification study) and control group (n = 10, pilot study; n = 30 verification study) and assessed by allele-discrimination real-time PCR to detect SNP c.355G>A. Using the allele-discrimination PCR, all of the samples are identified correctly with the genotype GG (wild-type, WT), GA (heterozygote, HET), or AA (homozygote, HOM) using the first analysis and no animals are not genotyped. We demonstrated that this novel method can be used to distinguish KD timely, accurately, and without ambiguity in HOM, WT, and HET animals. This protocol represents a great opportunity to increase accuracy and speed in KD research.

Krabbe disease (KD) is a lysosomal storage disorder characterized by severe neurodegeneration and demyelination. It is caused by mutations in the galactosylceramidase (GALC) gene, leading to the accumulation of psychosine, a neurotoxic metabolite. This study presents an optimized workflow for the production and characterization of recombinant murine GALC (rm‐GALC) from HEK293T cells, aiming to improve the feasibility of enzyme replacement therapy (ERT) for KD. An affinity chromatography protocol is refined to purify His‐tagged rm‐GALC, followed by buffer exchange and concentration steps to produce a stable and active enzyme suitable for subsequent in vitro applications. The purified rm‐GALC is characterized for enzymatic activity, purity, and stability using SDS‐PAGE, immunoblotting, and dynamic light scattering (DLS). In vitro assays reveal dose‐dependent enzymatic activity recovery in KD primary cells upon rm‐GALC administration, with no adverse effects on cell viability up to the physiological GALC dose. Additionally, GALC treatment at the physiological dose restored autophagic function in KD cells, as shown by LC3 and p62 marker analyses, confirming its compatibility with lysosomal‐autophagic pathways. Conversely, supra‐physiological GALC administration resulted in decreased viability and autophagy impairment. Finally, the feasibility of loading GALC into a polymeric nanovector based on stabilized reverse micelles is investigated. These findings highlight the critical importance of precise GALC dose regulation in developing a safe and effective enzyme replacement therapy (ERT) strategy for Krabbe disease (KD), further supporting the potential of a nanovector‐mediated ERT approach.

Enzyme aggregation and polymer encapsulation are complementary strategies used to overcome the intrinsic instability and limited bioavailability of hydrophilic enzymes in medical uses. In this work, we report a systematic study of polymer‐encapsulated cross‐linked enzyme aggregates (pe‐CLEAs) as a hierarchical nanostructured platform for the stabilization and delivery of galactosylceramidase (GALC), a lysosomal enzyme used in the enzyme replacement therapy for Krabbe disease. GALC‐CLEAs were produced via cross‐linking and controlled precipitation, yielding stable aggregates with small hydrodynamic diameters (40–66 nm) and narrow polydispersity index (PDI < 0.1). Scaling up the synthesis by 50‐fold markedly enhanced total enzymatic output, reaching 1225.4 ± 213.2 U·mL ⁻¹ . GALC‐CLEAs were subsequently encapsulated within PLGA nanoparticles. Systematic screening of enzyme‐to‐polymer ratios revealed an optimal formulation at 75 μg GALC per mg PLGA, displaying a maximum specific activity of 6.0 ± 1.3 U·mg ⁻¹ . Colloidal stability and retention assays showed that pe‐CLEAs preserved full catalytic activity and retained enzyme confinement over 30 days at 4°C. At 37°C, pe‐CLEAs exhibited time‐dependent size dynamics while retaining substantial activity. In vitro evaluations in primary fibroblasts derived from Twitcher mice (a Krabbe disease model) demonstrated complete cytocompatibility up to 5.0 mg·mL ⁻¹ and robust intracellular recovery of GALC activity across tested doses (0.5–5.0 mg·mL ⁻¹ ), outperforming equivalent doses of native enzyme. This work provides a comprehensive physicochemical and functional characterization of pe‐CLEAs, elucidating how hierarchical aggregation and polymer encapsulation can synergistically preserve stability and bioactivity. The design principles outlined here support the rational development of polymer–enzyme nanocarriers for enzyme replacement therapy and related biomedical applications.