RNA interference induced by small interfering RNA (siRNA) is a promising strategy for the treatment of various intractable diseases including cancer. Lipid nanoparticles (LNP) composed of ionizable lipids and siRNA are known as a leading siRNA delivery system. However, LNPs composed of conventional ionizable lipids will be aggregated in the physiological environment because of loss of ionization. Therefore, the inclusion of hydrophilic polymer-conjugated lipids such as polyethylene glycol (PEG)-conjugated lipid is required to improve the LNP stability. Herein, we synthesized a novel charge-reversible lipid derivative, dioleoylglycerophosphate-diethylenediamine conjugate (DOP-DEDA). The surface of LNP composed of DOP-DEDA (DOP-DEDA LNP) was constantly ionized and positively charged at pH 6.0, almost neutral at pH 7.4, and negatively charged at pH 8.0. Importantly, DOP-DEDA LNP were stable in the physiological milieu without PEG-conjugated lipid. DOP-DEDA LNP disrupted the red blood cells only under the low-pH condition in a hemolysis assay, suggesting that the interaction between DOP-DEDA LNP and biological membranes is pH-dependent. DOP-DEDA LNP encapsulating siRNA against polo-like kinase 1 (siPLK1) highly suppressed the expression of PLK1 mRNA and its protein. The cellular uptake of DOP-DEDA LNP was increased in an apolipoprotein E3 (apoE3) dose-dependent manner. In addition, DOP-DEDA LNP was taken up into cancer cells via both clathrin- and caveola-mediated endocytosis pathways. These findings indicate that LNP composed of this charge-reversible lipid should be a highly stable and potent siRNA delivery vector.

2019-03-20·ACS Applied Materials & Interfaces2区 · 材料科学

Lipid-mRNA Nanoparticle Designed to Enhance Intracellular Delivery Mediated by Shock Waves

2区 · 材料科学

Article

作者: Cleveland, Robin O. ; Shrivastava, Shamit ; Zhang, Jing ; Rabbitts, Terence H.

Cellular membranes are, in general, impermeable to macromolecules (herein referred to as macrodrugs, e.g., recombinant protein, expression plasmids, or mRNA), which is a major barrier for clinical translation of macrodrug-based therapies. Encapsulation of macromolecules in lipid nanoparticles (LNPs) can protect the therapeutic agent during transport through the body and facilitate the intracellular delivery via a fusion-based pathway. Furthermore, designing LNPs responsive to stimuli can make their delivery more localized, thus limiting the side effects. However, the principles and criteria for designing such nanoparticles remain unclear. We show that the thermodynamic state of the lipid membrane of the nanoparticle is a key design principle for acoustically responsive fusogenic nanoparticles. We have optimized a cationic LNP (designated LNP_LH) with two different phase transitions near physiological conditions for delivering mRNA. A bicistronic mRNA encoding a single domain intracellular antibody fragment and green fluorescent protein (GFP) was introduced into a range of human cancer cell types using LNP_LH, and the protein expression was measured via fluorescence corresponding to the GFP expression. The LNP_LH/mRNA complex demonstrated low toxicity and high delivery, which was significantly enhanced when the transfection occurred in the presence of acoustic shock waves. The results suggest that the thermodynamic state of LNPs provides an important criterion for stimulus responsive fusogenic nanoparticles to deliver macrodrugs to the inside of cells.

Pharmaceutics

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

Article

作者: Sato, Risa ; Oyama, Ryotaro ; Takeuchi, Hina ; Handa, Takumi ; Akita, Hidetaka ; Uchida, Yasuo ; Taira, Ryoga ; Gomi, Masaki ; Tezuka, Kenta ; Suzuki, Masayoshi ; Nishio, Kazuma ; Sakurai, Yu ; Nakai, Yuta ; Harada, Atsuki ; Watanabe, Himeka ; Hashimoto, Kohei ; Tange, Kota

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.

100 项与 VEGFA mRNA-LNP(Southeast University) 相关的药物交易

登录后查看更多信息