pH-Responsive Lipid Nanoparticles Achieve Efficient mRNA Transfection in Brain Capillary Endothelial Cells.

The blood-brain barrier (BBB), which is comprised of brain capillary endothelial cells, plays a pivotal role in the transport of drugs from the blood to the brain. Therefore, an analysis of proteins in the endothelial cells, such as transporters and tight junction proteins, which contribute to BBB function, is important for the development of therapeutics for the treatment of brain diseases. However, gene transfection into the vascular endothelial cells of the BBB is fraught with difficulties, even in vitro. We report herein on the development of lipid nanoparticles (LNPs), in which mRNA is encapsulated in a nano-sized capsule composed of a pH-activated and reductive environment-responsive lipid-like material (ssPalm). We evaluated the efficiency of mRNA delivery into non-polarized human brain capillary endothelial cells, hCMEC/D3 cells. The ssPalm LNPs permitted marker genes (GFP) to be transferred into nearly 100% of the cells, with low toxicity in higher concentration. A proteomic analysis indicated that the ssPalm-LNP had less effect on global cell signaling pathways than a Lipofectamine MessengerMAX/GFP-encoding mRNA complex (LFN), a commercially available transfection reagent, even at higher mRNA concentrations.

Proteomics-based investigation of cerebrovascular molecular mechanisms in cerebral amyloid angiopathy by the FFPE-LMD-PCT-SWATH method.

BACKGROUND: Cerebral amyloid angiopathy (CAA) occurs in 80% of patients with Alzheimer's disease (AD) and is mainly caused by the abnormal deposition of Aß in the walls of cerebral blood vessels. Cerebrovascular molecular mechanisms in CAA were investigated by using comprehensive and accurate quantitative proteomics. METHODS: Concerning the molecular mechanisms specific to CAA, formalin-fixed paraffin-embedded (FFPE) sections were prepared from patients having AD neuropathologic change (ADNC) with severe cortical Aß vascular deposition (ADNC +/CAA +), and from patients having ADNC without vascular deposition of Aß (ADNC +/CAA -; so called, AD). Cerebral cortical vessels were isolated from FFPE sections using laser microdissection (LMD), processed by pressure cycling technology (PCT), and applied to SWATH (sequential window acquisition of all theoretical fragment ion spectra) proteomics. RESULTS: The protein expression levels of 17 proteins in ADNC +/CAA +/H donors (ADNC +/CAA + donors with highly abundant Aß in capillaries) were significantly different from those in ADNC +/CAA - and ADNC -/CAA - donors. Furthermore, we identified 56 proteins showing more than a 1.5-fold difference in average expression levels between ADNC +/CAA + and ADNC -/CAA - donors, and were significantly correlated with the levels of Aß or Collagen alpha-2(VI) chain (COL6A2) (CAA markers) in 11 donors (6 ADNC +/CAA + and 5 ADNC -/CAA -). Over 70% of the 56 proteins showed ADNC +/CAA + specific changes in protein expression. The comparative analysis with brain parenchyma showed that more than 90% of the 56 proteins were vascular-specific pathological changes. A literature-based pathway analysis showed that 42 proteins are associated with fibrosis, oxidative stress and apoptosis. This included the increased expression of Heat shock protein HSP 90-alpha, CD44 antigen and Carbonic anhydrase 1 which are inhibited by potential drugs against CAA. CONCLUSIONS: The combination of LMD-based isolation of vessels from FFPE sections, PCT-assisted sample processing and SWATH analysis (FFPE-LMD-PCT-SWATH method) revealed for the first time the changes in the expression of many proteins that are involved in fibrosis, ROS production and cell death in ADNC +/CAA + (CAA patients) vessels. The findings reported herein would be useful for developing a better understanding of the pathology of CAA and for promoting the discovery and development of drugs and biomarkers for CAA.

Upregulation of ribosome complexes at the blood-brain barrier in Alzheimer's disease patients.

The cerebrovascular-specific molecular mechanism in Alzheimer's disease (AD) was investigated by employing comprehensive and accurate quantitative proteomics. Highly purified brain capillaries were isolated from cerebral gray and white matter of four AD and three control donors, and examined by SWATH (sequential window acquisition of all theoretical fragment ion spectra) proteomics. Of the 29 ribosomal proteins that were quantified, 28 (RPLP0, RPL4, RPL6, RPL7A, RPL8, RPL10A, RPL11, RPL12, RPL14, RPL15, RPL18, RPL23, RPL27, RPL27A, RPL31, RPL35A, RPS2, RPS3, RPS3A, RPS4X, RPS7, RPS8, RPS14, RPS16, RPS20, RPS24, RPS25, and RPSA) were significantly upregulated in AD patients. This upregulation of ribosomal protein expression occurred only in brain capillaries and not in brain parenchyma. The protein expression of protein processing and N-glycosylation-related proteins in the endoplasmic reticulum (DDOST, STT3A, MOGS, GANAB, RPN1, RPN2, SEC61B, UGGT1, LMAN2, and SSR4) were also upregulated in AD brain capillaries and was correlated with the expression of ribosomal proteins. The findings reported herein indicate that the ribosome complex, the subsequent protein processing and N-glycosylation-related processes are significantly and specifically upregulated in the brain capillaries of AD patients.