The size of these particles ranged from 119 to 184 nm with PDI <0.3. 17 Particle properties, including particle size, zeta potential, and entrapment efficiency of Cas9 mRNA, were measured using a Zetasizer Nano ZS and a ribogreen assay ( Figures 1A,B and S1). According to the methods reported previously, 17– 22, 31 newly synthesized amino-ester-derived lipidlike compounds were formulated with 2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol), 1,2-dimyristoyl- sn-glycerol, methoxypoly(ethylene glycol) (DMG-PEG 2000), and Cas9 mRNA in an optimized molar ratio (lipid/DOPE/Chol/DMG-PEG 2000 = 20/30/40/0.75). 19, 22 The structures of the products were validated by 1H NMR spectroscopy and mass spectrometry ( Supporting Information).
MPA 20 DMG SERIES
Epoxide ( E) and acrylate ( O) series were synthesized according to the procedures reported previously. Moreover, we are capable of tuning the hydrolysis rate by incorporation of functional groups with different steric effects ( Scheme 1B).įirst, we installed lipid chains A and Ab on the four amines through a reductive amination reaction to afford amino-ester-derived lipidlike compounds ( Scheme 1A). We hypothesized that A and Ab series lipidlike compounds are biodegradable through interaction with esterases. The epoxide ( E) and acrylate ( O) series were reported previously for siRNA delivery, 19, 22 serving as control groups. The linear and branched ester series are termed as 9-oxononanoic acid ( Z)-non-2-en-yl ester ( A) and 9-oxononanoic acid 2-ethyl-hexane-1-yl ester ( Ab), respectively. As shown in Scheme 1, we installed four types of lipid chains, including two ester chains, one epoxide chain, and one acrylate chain on four amino cores, termed as N, N′-dimethyl-1,3-propanediamine ( DMPA), N-methyl-1,3-propanediamine ( MPA), 1,3-propanediamine ( PA), and N-(2-aminoethyl)-1,3-propanediamine ( AEPA). We report the design, synthesis, and biological evaluation of biodegradable amino-ester-derived LLNs for the delivery of Cas9 mRNA. 30 Hence, it is essential to develop new materials with high delivery efficiency and compatible biodegradability. 22, 29 For example, the biodegradable lipids reported previously were well-tolerated at a dose of 10 mg/kg in mice, whereas nonbiodegradable lipids caused acute mortality at a similar dose. Previous studies reported that biodegradable bonds enabled rapid elimination of lipids from plasma and tissues and substantially improved their tolerability in preclinical studies. 17, 20, 25– 28 Yet, many lipidlike compounds are not considered biodegradable, which may lead to potential side effects. 17– 24 Recently, a few LLN-based delivery systems demonstrated efficient delivery of mRNAs for expression of functional proteins, including factor IX, erythropoietin, and Cas9. 12– 16 Particularly, lipid and lipidlike nanoparticles (LNPs and LLNs) are representative biomaterials for efficient delivery of RNAs, including siRNA, miRNA, and mRNA. Nanomaterials have shown great promise for delivery of diverse therapeutic and diagnostic payloads. 7 Hence, new biomaterials are needed to overcome these obstacles and to enable broad applications of CRISPR/Cas9. 11 One alternative approach is to express Cas9 protein using Cas9 mRNA however, efficient delivery of Cas9 mRNA is formidable due to its large size (up to 4.5 k nucleotides), high density of negative charges, and weak tolerance of enzymes in serum. 6– 10 Yet, potential off-target effects are significant safety issues if Cas9 is present for a long time. 1– 5 Adeno-associated virus-derived vectors and DNA nanoclews were reported to deliver plasmid-encoding CRISPR/Cas in a wide variety of cells and animal models. CRISPR/Cas9-mediated genome editing has been widely used as a powerful tool for biological and therapeutic applications.
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