Liquid–liquid phase separation
(LLPS) is a crucial phenomenon
for the formation of functional membraneless organelles. However,
LLPS is also responsible for protein aggregation in various neurodegenerative
diseases such as amyotrophic lateral sclerosis, Alzheimer’s
disease, and Parkinson’s disease (PD). Recently, several reports,
including ours, have shown that α-synuclein (α-Syn) undergoes
LLPS and a subsequent liquid-to-solid phase transition, which leads
to amyloid fibril formation. However, how the environmental (and experimental)
parameters modulate the α-Syn LLPS remains elusive. Here, we
show that in vitro α-Syn LLPS is strongly dependent on the presence
of salts, which allows charge neutralization at both terminal segments
of protein and therefore promotes hydrophobic interactions supportive
for LLPS. Using various purification methods and experimental conditions,
we showed, depending upon conditions, α-Syn undergoes either
spontaneous (instantaneous) or delayed LLPS. Furthermore, we delineate
that the kinetics of liquid droplet formation (i.e., the critical
concentration and critical time) is relative and can be modulated
by the salt/counterion concentration, pH, presence of surface, PD-associated
multivalent cations, and N-terminal acetylation, which are all known
to regulate α-Syn aggregation in vitro. Together, our observations
suggest that α-Syn LLPS and subsequent liquid-to-solid phase
transition could be pathological, which can be triggered only under
disease-associated conditions (high critical concentration and/or
conditions promoting α-Syn self-assembly). This study will significantly
improve our understanding of the molecular mechanisms of α-Syn
LLPS and the liquid-to-solid transition.
First‐pass hepatic metabolism can significantly limit oral drug bioavailability. Drug transport from the intestine through the lymphatic system, rather than the portal vein, circumvents first‐pass metabolism. However, the majority of drugs do not have the requisite physicochemical properties to facilitate lymphatic access. Herein, we describe a prodrug strategy that promotes selective transport through the intestinal lymph vessels and subsequent release of drug in the systemic circulation, thereby enhancing oral bioavailability. Using testosterone (TST) as a model high first‐pass drug, glyceride‐mimetic prodrugs incorporating self‐immolative (SI) spacers, resulted in remarkable increases (up to 90‐fold) in TST plasma exposure when compared to the current commercial product testosterone undecanoate (TU). This approach opens new opportunities for the effective development of drugs where oral delivery is limited by first‐pass metabolism and provides a new avenue to enhance drug targeting to intestinal lymphoid tissue.
Background
The purpose of this study was to develop protein-/peptide-loaded nanoparticle-based delivery system, which can efficiently deliver therapeutic molecules to the lung via pulmonary delivery. The chitosan nanoparticles were prepared by the ionic gelation method, and bovine serum albumin was used as a model protein. These nanoparticles were characterized for size, zeta potential, encapsulation efficiency, cell cytotoxicity, uptake study, release profile and size distribution and uniformity. The chemical interaction of chitosan and protein was studied by XRD and FTIR. The integrity assessment of encapsulated protein into nanoparticle was studied by native and SDS-PAGE gel electrophoresis.
Results
The size and zeta potential of BSA nanoparticles were 193.53 ± 44.97 to 336.36 ± 94.63 and 12.73 ± 0.41 to 18.33 ± 0.96, respectively, with PDI values of 0.35–0.45. The encapsulation efficiency was in the range of 80.73 ± 6.37% to 92.34 ± 1.72%. The cumulative release of the BSA from the nanoparticles was 72.56 ± 6.67% in 2 weeks. The BSA-loaded nanoparticles showed good uptake and no significant cytotoxicity observed into the A549 cell line. In this study, it was also observed that during nanoparticles’ synthesis protein structure and integrity is not compromised. The nanoparticles showed controlled and sustained release with initial burst release. In TEM images, it was shown that nanoparticles’ distribution is uniform within nanometre range.
Conclusion
From this study, it was concluded that nanoparticles prepared by this method are suitable to deliver protein/peptide into the cells without any degradation of protein during process of nanoparticle fabrication.
Mastitis is the most devastating economic disease in dairy cattle. Mastitis in dairy cattle frequently occurs during the dry period or during early lactation. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus)are the main causative agents of mastitis in India. S. aureus can form microabscesses in the udder and develop a subclinical form of mastitis. This bacterial property hinders an effective cure during the lactation period. Antimicrobials used for treatments have a short half-life at the site of action because of frequent milking; thereforethey are unable to maintain the desired drug concentration for effective clearance of bacteria. We demonstrated the potential of ciprofloxacin-encapsulated nanocarriersthat can improve the availability of drugs and provide an effective means for mastitis treatment. These drug-loaded nanoparticles show low toxicity and slow clearance from the site of action. Antimicrobial activity against clinical strains of E. coli and S. aureus showed that the zone of inhibition depended on the dose (0.5 mg to 2 mg/mL nanoparticle solution from 11.6 to 14.5 mm and 15 to 18 mm). These nanoparticles showed good antimicrobial activity in broth culture and agar diffusion assay against bacteria.
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