Dataset Information

The histidine-rich peptide LAH4-L1 strongly promotes PAMAM-mediated transfection at low nitrogen to phosphorus ratios in the presence of serum.

ABSTRACT: Non-viral vectors are widely used and investigated for the delivery of genetic material into cells. However, gene delivery barriers like lysosomal degradation, serum inhibition and transient gene expression so far still limit their clinical applications. Aiming to overcome these limitations, a pH-sensitive hybrid gene vector (PSL complex) was designed by self-assembly of poly(amidoamine) (PAMAM) dendrimers, the histidine-rich peptide LAH4-L1 and the sleeping beauty transposon system (SB transposon system, a plasmid system capable of efficient and precise genomic insertion). Transfection studies revealed that PSL complexes achieved excellent efficiency in all investigated cell lines (higher than 90% in HeLa cells and over 30% in MDCK cells, a difficult-to-transfect cell line). Additionally, the PSL complexes showed high serum tolerance and exhibited outstanding transfection efficiency even in medium containing 50% serum (higher than 90% in HeLa cells). Moreover, a high level of long-term gene expression (over 30% in HeLa cells) was observed. Furthermore, PSL complexes not only resulted in high endocytosis, but also showed enhanced ability of endosomal escape compared to PAMAM/DNA complexes. These results demonstrate that simple association of PAMAM dendrimers, LAH4-L1 peptides and the SB transposon system by self-assembly is a general and promising strategy for efficient and safe gene delivery.

SUBMITTER: Liu N

PROVIDER: S-EPMC5575053 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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The histidine-rich peptide LAH4-L1 strongly promotes PAMAM-mediated transfection at low nitrogen to phosphorus ratios in the presence of serum.

Liu Nan N Bechinger Burkhard B Süss Regine R

Scientific reports 20170829 1

Non-viral vectors are widely used and investigated for the delivery of genetic material into cells. However, gene delivery barriers like lysosomal degradation, serum inhibition and transient gene expression so far still limit their clinical applications. Aiming to overcome these limitations, a pH-sensitive hybrid gene vector (PSL complex) was designed by self-assembly of poly(amidoamine) (PAMAM) dendrimers, the histidine-rich peptide LAH4-L1 and the sleeping beauty transposon system (SB transpos ...[more]

PMID: 28852016

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Project description:The histidine-rich designer peptide LAH4-L1 exhibits antimicrobial and potent cell-penetrating activities for a wide variety of cargo including nucleic acids, polypeptides, adeno-associated viruses, and nanodots. The non-covalent complexes formed between the peptide and cargo enter the cell via an endosomal pathway where the pH changes from neutral to acidic. Here, we investigated the membrane interactions of the peptide with phospholipid bilayers and its membrane topology using static solid-state NMR spectroscopy. Oriented 15N solid-state NMR indicates that in membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) 3:1 mol/mole and at neutral pH, the peptide adopts transmembrane topologies. Furthermore, 31P and 2H solid-state NMR spectra show that liquid crystalline 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and POPC/POPS 3:1 liposomes retain a bilayer macroscopic phase even at the highest peptide concentrations investigated, with an oblate orientational distribution of the phospholipids at a peptide/lipid ratio of 1:5. At pH 5, as it occurs in the endosome, the alignment of LAH4-L1 at a peptide/lipid ratio of 1:25 is predominantly parallel to POPC/POPS 3:1 bilayers (prolate deformation) when at the same time it induces a considerable decrease of the deuterium order parameter of POPC/2H31-POPS 3:1. In addition, when studied in mechanically supported lipid membranes, a pronounced disordering of the phospholipid alignment is observed. In the presence of even higher peptide concentrations, lipid spectra are observed that suggest the formation of magnetically oriented or isotropic bicelles. This membrane-disruptive effect is enhanced for gel phase DMPC membranes. By protonation of the four histidines in acidic environments, the overall charge and hydrophobic moment of LAH4-L1 considerably change, and much of the peptide is released from the cargo. Thus, the amphipathic peptide sequences become available to disrupt the endosomal membrane and to assure highly efficient release from this organelle.

Project description:The ecological stoichiometric characteristics of soil elements have greatly enhanced our understanding of the circulation of soil nutrients. However, there is limited knowledge regarding the alteration of carbon, nitrogen, and phosphorus stoichiometric ratios in deep soil after afforestation. To examine the variations in stoichiometric ratios of soil elements with different vegetation types, restoration times, and soil depths, we collected soil samples from grassland, Caragana korshinskii shrubland, and Picea asperata forestland at different stand ages (10a, 25a, and 40a) in Xining City, which is located on the Loess Plateau. Our results showed that, at 25a, the carbon-to-nitrogen (C:N) and carbon-to-phosphorus (C:P) ratios were significantly higher in the grassland soil than under other vegetation types, whereas the nitrogen-to-phosphorus (N:P) ratio had no significant difference among the three vegetation types. At 40a, the ratios of soil C:N, C:P, and N:P in the shrubland were the highest. With the increasing of the restoration time, the ratios of soil C:N, C:P, and N:P in grassland with 25a became higher than for 40a or 10a. The ratios in the shrubland were highest at 40a, followed by 25a and then 10a, while the ratios in the forestland showed no significant difference. At 40a, the soil C:N, C:P, and N:P ratios of shrubland were highest at the soil depth of 40-100 cm. The soil C:N, C:P, and N:P ratios showed positive correlations with soil ammonium nitrogen and nitrate nitrogen, and the soil N:P ratios showed a negative correlation with soil available phosphorus. Plant diversity significantly influenced the soil stoichiometric ratio of the upper soil layer. In the upper soil layer (0-40 cm), species richness showed a positive correlation with soil C:N, C:P, and N:P ratios, and the Margalef index exhibited a positive correlation with soil C:N and C:P ratios. The results of this study indicate that the stoichiometric ratio and nutrient availability of Caragana korshinskii shrubland were the highest over time. Therefore, these findings can be served as a valuable reference for local revegetation and ecological restoration.

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Dataset Information

The histidine-rich peptide LAH4-L1 strongly promotes PAMAM-mediated transfection at low nitrogen to phosphorus ratios in the presence of serum.

Publications

The histidine-rich peptide LAH4-L1 strongly promotes PAMAM-mediated transfection at low nitrogen to phosphorus ratios in the presence of serum.

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