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.

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.

Regional Differences in the Absolute Abundance of Transporters, Receptors and Tight Junction Molecules at the Blood-Arachnoid Barrier and Blood-Spinal Cord Barrier among Cervical, Thoracic and Lumbar Spines in Dogs.

PURPOSE: The purpose of the present study was to quantitatively determine the expression of transporters, receptors and tight junction molecules at the blood-arachnoid barrier (BAB) and blood-spinal cord barrier (BSCB) in cervical, thoracic and lumbar spines from dogs. METHODS: The expression levels of 31 transporters, 3 receptors, 1 tight junction protein, and 3 marker proteins in leptomeninges and capillaries isolated from spines (3 male and 2 female dogs) were determined by quantitative Targeted Absolute Proteomics (qTAP). The units were converted from fmol/µg protein to pmol/cm (absolute abundance at the BAB and the BSCB in a 1 cm section of spine). RESULTS: The expression of MDR1 and BCRP were greater at the BSCB compared to the BAB (especially in the cervical cord), and the expressions at the lumbar BSCB were lower than that for the cervical BSCB. Among the organic anionic and cationic drug transporters, OAT1, OAT3, MRP1, OCT2 and MATE1/2 were detected only in the BAB, and not at the BSCB). The expression of these transporters was higher in the order: lumbar > thoracic > cervical BAB. The expressions of GLUT1, 4F2hc, EAAT1, 2, PEPT2, CTL1, and MCT1 at the BSCB of the cervical cord were higher than the corresponding values for the cervical BAB, and these values decreased in going down the spinal cord. CONCLUSION: These results provide a better understanding of the molecular mechanisms underlying the concentration gradients of drugs and endogenous substances in the cerebrospinal fluid and parenchyma of the spinal cord.

A human blood-arachnoid barrier atlas of transporters, receptors, enzymes, and tight junction and marker proteins: Comparison with dog and pig in absolute abundance.

The purpose of this study was to elucidate the absolute abundance of transporters, enzymes, receptors, and tight junction and marker proteins at human blood-arachnoid barrier (BAB) and compare with those of dogs and pigs. Protein expression levels in plasma membrane fractions of brain leptomeninges were determined by quantitative targeted absolute proteomics. To realistically compare the absolute abundance of target molecules at the BAB among humans, dogs, and pigs, the unit was converted from fmol/µg-protein to pmol/cm2 -leptomeninges. Of a total of 70 proteins, 52 were detected. OAT1, OAT3, GLUT1, 4F2hc, EAAT1, EAAT2, MCT8, SMVT, CTL2, GFAP, Claudin-5, Na+ /K+ -ATPase, COMT, GSTP1, and CES1 were abundantly expressed at the human BAB (>1 pmol/cm2 ). The protein expression levels were within a 3-fold difference for 16 out of 33 proteins between humans and dogs and for 13 out of 28 proteins between humans and pigs. Both human-dog and human-pig differences in protein expression levels were within 3-fold for OAT1, OAT3, 4F2hc, xCT, OCT2, MDR1, BCRP, PEPT2, SYP, and MCT1. In contrast, OCT3, MCT4, and OATP1A2 were detected in humans but not in dogs or pigs. MRP3 was detected in dogs and pigs but not in humans. The absolute level of GLUT1 in humans was nearly the same as that in dogs but was 6.14-fold greater in pigs. No significant differences in the levels were observed between male and female dogs for nearly all molecules. These results should be helpful in understanding the physiological roles of BAB and cerebrospinal fluid pharmacokinetics in humans and their differences from dogs and pigs.

Abundant Expression of OCT2, MATE1, OAT1, OAT3, PEPT2, BCRP, MDR1, and xCT Transporters in Blood-Arachnoid Barrier of Pig and Polarized Localizations at CSF- and Blood-Facing Plasma Membranes.

The physiologic and pharmacologic roles of the blood-arachnoid barrier (BAB) remain unclear. Therefore, the purpose of the present study was to comprehensively evaluate and compare the absolute protein expression levels of transporters in the leptomeninges and plexus per cerebrum, and to determine the localizations of transporters at the cerebrospinal fluid (CSF)-facing and blood (dura)-facing plasma membranes of the BAB in pig. Using multidrug resistance protein 1 (MDR1) and organic anion transporter (OAT) 1 as blood (dura)-facing and CSF-facing plasma membrane marker proteins, respectively, we established that breast cancer resistance protein (BCRP), multidrug resistance-associated protein (MRP) 4, organic anion-transporting polypeptide (OATP) 2B1, multidrug and toxin extrusion protein 1 (MATE1), and glucose transporter 1 (GLUT1) are localized at the blood-facing plasma membrane, and OAT3, peptide transporter (PEPT) 2, MRP3, organic cation transporter (OCT) 2, xCT, monocarboxylate transporter (MCT) 1, MCT4, and MCT8 are localized at the CSF-facing plasma membrane of the BAB. The absolute protein expression levels of OAT1, OAT3, MDR1, BCRP, PEPT2, xCT, MATE1, OCT2, and 4f2hc in the whole BAB surrounding the entire cerebrum were much larger than those in the total of the choroid plexuses forming the blood-cerebrospinal fluid barrier (BCSFB). Although MRP4, OATP2B1, MCT8, GLUT1, and MCT1 were also statistically significantly more abundant in the BAB than in the choroid plexuses per porcine cerebrum, these transporters were nevertheless almost equally distributed between the two barriers. In contrast, OATP1A2, MRP1, OATP3A1, and OCTN2 were specifically expressed in the choroid plexus. These results should be helpful in understanding the relative overall importance of transport at the BAB compared with that at the BCSFB, as well as the rank order of transport capacities among different transporters at the BAB, and the directions of transport mediated by individual transporters. SIGNIFICANCE STATEMENT: We found that BCRP, MRP4, OATP2B1, MATE1, and GLUT1 localize at the blood-facing plasma membrane of the blood-arachnoid barrier (BAB), while OAT3, PEPT2, MRP3, OCT2, xCT, MCT1, MCT4, and MCT8 localize at the CSF-facing plasma membrane. 4F2hc is expressed in both membranes. For OAT1, OAT3, MDR1, BCRP, PEPT2, xCT, MATE1, OCT2, and 4f2hc, the absolute protein expression levels in the whole BAB surrounding the entire cerebrum are much greater than the total amounts in the choroid plexuses.