Size-dependent specific targeting and efficient gene silencing in peritoneal macrophages using a pH-sensitive cationic liposomal siRNA carrier

Matsui

Journal of Controlled Release

et al. 2018

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Lipid nanoparticles (LNPs) are one of the leading technologies for the in vivo delivery of short interfering RNA (siRNA). Cationic lipids are an important component for efficient endosomal escape via membrane fusion followed by release of siRNAs in cytosol where the site of action is located. A high cationic lipid/siRNA charge ratio is usually necessary for maximizing the gene silencing activity of the siRNA-loaded LNPs. However, high levels of cationic lipids are known to cause cytotoxicity through interactions with negatively charged biocomponents. A strategy for solving this dilemma is important, in terms of producing clinically applicable LNPs with a wide therapeutic window. We herein report on the development of LNPs with a high gene silencing activity and a low cationic lipid/siRNA charge ratio, which we refer to as low lipid core-nanoparticles (LLC-NPs). The negative charges of the siRNAs were neutralized by protamines, cationic proteins, to reduce the net dose of cationic lipid, YSK05, which was developed in our laboratory, for endosomal escape, resulting in preserved fusogenic activity and gene silencing activity, both in vitro and in vivo factor VII mouse model. In addition, the LLC-NPs showed an improved hepatotoxicity compared to conventional LNPs, which have a relatively higher cationic lipid/siRNA charge ratio. The concept of the LLC-NPs helps to realize clinically applicable LNPs with a wide therapeutic window and has the potential for use in various applications and for the delivery of different classes of nucleic acid.

Section: Toxicity Of the Lnps In Vivomentioning

confidence: 86%

Section: Evaluation Of Biodistibution and Elimination From Livermentioning

confidence: 99%

Neutralization of negative charges of siRNA results in improved safety and efficient gene silencing activity of lipid nanoparticles loaded with high levels of siRNA

Matsui

Journal of Controlled Release

et al. 2018

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“…The LNPs were prepared by a tertiary butanol (t-BuOH) dilution procedure, as previously reported. 33,37,38 The lipids, which include 1050 nmol of YSK lipids (YSK05 or YSK12-C4 or a mixture of both), 450 nmol of cholesterol, and 30 nmol of m PEG 2k -DMG -which represent a molar ratio of 70:30:2, respectively -were first dissolved in 400 µL of 90% (v/v) t-BuOH. When a fluorescent material (DiI or DiD) was incorporated into the LNPs, it was added at a concentration of 0.5-1 mol% or 0.15 mol% (of the total lipid) to the lipid solution.…”

Section: Preparation Of Lnpsmentioning

confidence: 99%

“…We previously reported on the development of our original LNPs, which can be prepared using several types of lipids that were synthesized in our laboratory, and are referred to as YSK lipids, to deliver siRNA to cells, both in vitro and in vivo. [32][33][34][35][36][37][38] LNPs, especially those containing lipids, namely YSK05 and YSK13-C3, showed a high cellular uptake, strong endosomal escape, and efficient siRNA delivery and silencing activity, especially in hepatocytes. 33,34 We also found that the pKa value of the LNP membrane influences its intrahepatic distribution.…”

mentioning

confidence: 99%

Mixing lipids to manipulate the ionization status of lipid nanoparticles for specific tissue targeting

Shobaki

Harashima

2018

IJN

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IntroductionThe development of targeted drug delivery systems is a rapidly growing area in the field of nanomedicine.MethodsWe report herein on optimizing the targeting efficiency of a lipid nanoparticle (LNP) by manipulating the acid dissociation constant (pKa) value of its membrane, which reflects its ionization status. Instead of changing the chemical structure of the lipids to achieve this, we used a mixture of two types of pH-sensitive cationic lipids that show different pKa values in a single LNP. We mixed various ratios of YSK05 and YSK12-C4 lipids, which have pKa values of 6.50 and 8.00, respectively, in one formulation (referred to as YSK05/12-LNP).ResultsThe pKa of the YSK05/12-LNP was dependent not only on the molar ratio of each lipid but also on the individual contribution of each lipid to the final pKa (the YSK12-C4 lipid showed a higher contribution). Furthermore, we succeeded in targeting and delivering short interfering RNA to liver sinusoidal endothelial cells using one of the YSK05/12-LNPs which showed an optimum pKa value of 7.15 and an appropriate ionization status (~36% cationic charge) to permit the particles to be taken up by liver sinusoidal endothelial cells.ConclusionThis strategy has the potential for preparing custom LNPs with endless varieties of structures and final pKa values, and would have poten tial applications in drug delivery and ionic-based tissue targeting.

Journal of Controlled Release

“…On the other hand, because YSK05 is neutral at physiological pH 7.4, a YSK05-containing MEND (YSK-MEND) would have a high biocompatibility. These characteristic properties of the YSK-MEND enabled us to inhibit mRNA expression, even in in vivo situations, such as the murine liver [25][26][27][28], a humanized chimeric liver infected with human hepatitis virus B and C [29,30], liver sinusoidal endothelial cells [31], brain endothelial cells [32], peritoneal macrophages [33] and cancer cells [34,35]. For tumor endothelial cell (TEC)-targeting, we modified the YSK-MEND with a cyclic RGD peptide (RGD-MEND), which is known to be a ligand against the α V β 3 integrin heterodimer, which is highly expressed in TECs and some types of cancer cells [36].…”

Section: Introductionmentioning

confidence: 99%

Modality of tumor endothelial VEGFR2 silencing-mediated improvement in intratumoral distribution of lipid nanoparticles

Yamamoto

Kato

Sakurai

et al. 2017

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The vascular endothelial growth factor (VEGF)-mediated enhancement in vascular permeability is considered to be a major factor in tumor-targeting delivery via the enhanced permeability and retention (EPR) effect. We previously reported that the silencing of the endothelial VEGF receptor (VEGFR2) by a liposomal siRNA system (RGD-MEND) resulted in an enhanced intratumoral distribution of polyethylene glycol (PEG)-modified liposomes (LPs) in a renal cell carcinoma, a type of hypervascularized cancer, although the inhibition of VEGF signaling would be expected to decrease the permeability of the tumor vasculature. We herein report that the enhancement in the intratumoral distribution of LPs by VEGFR2 inhibition was dependent on the vascular type of the tumor (stroma vessel type; SV and tumor vessel type; TV). In the case of TV-type tumors (renal cell carcinoma and hepatocellular carcinoma), inhibiting VEGFR2 improved intratumoral distribution, while no effect was found in the case of SV-type tumors (colorectal cancer). Moreover, through a comparison of the intratumoral distribution of LPs with a variety of physical properties (100nm vs 400nm, neutral vs negative vs positive), VEGFR2 inhibition was found to alter the tumor microenvironment, including heparan sulfate proteoglycans (HSPGs). In addition, the results regarding the effect of the size of nanoparticles indicated that VEGFR2 inhibition improved the penetration of nanoparticles through the vessel wall, but not via permeability, suggesting the involvement of an unknown mechanism. Our findings suggest that a combination of anti-angiogenic therapy and delivery via the EPR effect would be useful in certain cases, and that altering the tumor microenvironment by VEGFR2 blockade has a drastic effect on the intratumoral distribution of nanoparticles.