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Constrained Nanoparticles Deliver siRNA and sgRNA to T Cells In Vivo without Targeting Ligands.

ABSTRACT: T cells help regulate immunity, which makes them an important target for RNA therapies. While nanoparticles carrying RNA have been directed to T cells in vivo using protein- and aptamer-based targeting ligands, systemic delivery to T cells without targeting ligands remains challenging. Given that T cells endocytose lipoprotein particles and enveloped viruses, two natural systems with structures that can be similar to lipid nanoparticles (LNPs), it is hypothesized that LNPs devoid of targeting ligands can deliver RNA to T cells in vivo. To test this hypothesis, the delivery of siRNA to 9 cell types in vivo by 168 nanoparticles using a novel siGFP-based barcoding system and bioinformatics is quantified. It is found that nanomaterials containing conformationally constrained lipids form stable LNPs, herein named constrained lipid nanoparticles (cLNPs). cLNPs deliver siRNA and sgRNA to T cells at doses as low as 0.5 mg kg-1 and, unlike previously reported LNPs, do not preferentially target hepatocytes. Delivery occurs via a chemical composition-dependent, size-independent mechanism. These data suggest that the degree to which lipids are constrained alters nanoparticle targeting, and also suggest that natural lipid trafficking pathways can promote T cell delivery, offering an alternative to active targeting approaches.

SUBMITTER: Lokugamage MP

PROVIDER: S-EPMC6819129 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Constrained Nanoparticles Deliver siRNA and sgRNA to T Cells In Vivo without Targeting Ligands.

Lokugamage Melissa P MP Sago Cory D CD Gan Zubao Z Krupczak Brandon R BR Dahlman James E JE

Advanced materials (Deerfield Beach, Fla.) 20190829 41

T cells help regulate immunity, which makes them an important target for RNA therapies. While nanoparticles carrying RNA have been directed to T cells in vivo using protein- and aptamer-based targeting ligands, systemic delivery to T cells without targeting ligands remains challenging. Given that T cells endocytose lipoprotein particles and enveloped viruses, two natural systems with structures that can be similar to lipid nanoparticles (LNPs), it is hypothesized that LNPs devoid of targeting li ...[more]

PMID: 31465135

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Project description:Cancer is one of the top-ranked noncommunicable diseases causing deaths to nine million people and affecting almost double worldwide in 2018. Tremendous advancement in surgery, chemotherapy, radiation and targeted immunotherapy have improved the rate of cure and disease-free survival. As genetic mutations vary in different cancers, potential of customized treatment to silence the problem gene/s at the translational level is being explored too. Yet delivering therapeutics at the required dosage only to the affected cells without affecting the healthy ones, is a big hurdle to be overcome. Scientists worldwide have been working to invent a smart drug delivery system for targeted delivery of therapeutics to tumor tissues only. As part of such an effort, few organic nanocarriers went to clinical trials, while inorganic nanoparticles (NPs) are still in development stage despite their many customizable properties. Carbonate apatite (CA), a pH sensitive nanocarrier has emerged as an efficient delivery system for drugs, plasmids and siRNAs in preclinical models of breast and colon cancers. Like hydroxyapatite (HA) which serves as a classical tool for delivery of genetic materials such as siRNA and plasmid, CA is an apatite-based synthetic carrier. We developed simplified methods of formulating CA-in-DMEM and a DMEM-mimicking buffer and HA in a HEPES-buffered solution and characterized them in terms of size, stability, protein corona (PC) composition, cytotoxicity, siRNA delivery efficiency in breast cancer cells and siRNA biodistribution profile in a mouse model of breast cancer. Particle growth was analyzed via spectrophotometry and light microscopy, size was measured via dynamic light scattering and scanning electron microscopy and confirmation of functional groups in apatite structures was made by FT-IR. siRNA-binding was analyzed via spectrophotometry. Stability of the formulation solutions/buffers was tested over various time points and at different temperatures to determine their compatibility in the context of practical usage. Cellular uptake was studied via fluorescence microscopy. MTT assay was performed to measure the cytotoxicity of the NPs. Liquid chromatography-mass spectrometry was carried out to analyze the PC formed around all three different NPs in serum-containing media. To explore biodistribution of all the formulations, fluorescence-labeled siRNA-loaded NPs were administered intravenously prior to analysis of fluorescence intensity in the collected organs and tumors of the treated mice. The size of NPs in 10% serum-containing media was dramatically different where CA-in-DMB and HA were much larger than CA-in-DMEM. Effect of media was notable on the PC composition of all three NPs. All three NPs bound albumin and some common protease inhibitors involved in bone metabolism due to their compositional similarity to our bone materials. Moreover, CA also bound heme-binding proteins and opsonins. Unlike CA, HA bound different kinds of keratins. Difference in PC constitution was likely to influence accumulation of NPs in various organs including those of reticuloendothelial system, such as liver and spleen and the tumor. We found 10 times more tumor accumulation of CA-in-DMB than CA-in-DMEM, which could be due to more stable siRNA-binding and distinct PC composition of the former. As a nanocarrier CA is more efficient than HA for siRNA delivery to the tumor. CA prepared in a buffer containing only the mere constituents was potentially more efficient than classical CA prepared in DMEM, owing to the exclusion of interference attributed by the inorganic ions and organic molecules present in DMEM.

Dataset Information

Constrained Nanoparticles Deliver siRNA and sgRNA to T Cells In Vivo without Targeting Ligands.

Publications

Constrained Nanoparticles Deliver siRNA and sgRNA to T Cells In Vivo without Targeting Ligands.

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