Journal: ACS nano

Article Title: Ionizable lipid nanoparticles for therapeutic base editing of congenital brain disease

doi: 10.1021/acsnano.3c02268

Figure Lengend Snippet: (A) Chemical structures of epoxide-terminated alkyl tails (green box) and polyamine cores (blue box) combined to generate an ionizable lipid library. Formulation of LNPs via microfluidic mixing with an ethanol phase containing ionizable lipid, PEG-lipid, cholesterol, and DOPE and an aqueous phase containing luciferase mRNA is also visualized. LNPs were named based on the alkyl tail length (A-C) and polyamine core (1–4) of the ionizable lipid incorporated into the formulation. (B) Size and PDI of each LNP formulation in the LNP library. (C) Zeta potential (radius of circle), pKa (gradient color), and encapsulation efficiency (centered number) for each LNP formulation. (D) Scheme demonstrating LNP screening in E18 BALB/c fetuses or P0 BALB/c neonates via ICV injection. (E) IVIS imaging showing luciferase expression in a representative C3 LNP-treated fetus (left) and neonate (right) relative to PBS-treated controls. (F) Quantification of luciferase signal from fetal brains treated with each LNP. (G) Quantification of luciferase signal from the neonatal brains treated with a subset of LNPs. All luminescence readings are represented as normalized total flux. ** p < 0.01, **** p < 0.0001 by one-way analysis of variance (ANOVA) with post-hoc Dunnett’s test compared to MC3 (fetus) and C3 (neonate). Outliers were detected using Grubbs’ test and removed from analysis; minimum n = 3 per treatment group; error bars represent SEM.

Article Snippet: LNP formulation Ionizable lipids, prepared as described above, or DLin-MC3-DMA (MedChem Express) were combined in an ethanol phase with cholesterol (Sigma-Aldrich), DOPE (Avanti), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (C14-PEG2000, Avanti) at a molar ratio of 35:46.5:16:2.5 or at molar ratios specified in Fig. S6 .

Techniques: Formulation, Luciferase, Zeta Potential Analyzer, Encapsulation, Injection, Imaging, Expressing

Journal: ACS nano

Article Title: Ionizable lipid nanoparticles for therapeutic base editing of congenital brain disease

doi: 10.1021/acsnano.3c02268

Figure Lengend Snippet: (A) Scheme demonstrating the three LNP formulation parameters evaluated: 1) excipient molar ratios, 2) N:P ratio, and 3) ABE to sgRNA mass ratio. (B) Normalized mean fluorescence intensity (MFI) in Neuro-2a cells after treatment with the C3 LNP DOE library relative to the original C3 LNP formulation (dotted line). (C) Normalized MFI in Neuro-2a cells after treatment with C3 LNPs formulated at a range of N:P ratios. (D) Quantification of luminescence signal (normalized total flux) from brains of neonates treated with C3 LNPs prepared at the top-performing N:P ratios in vitro. (E) Sequencing results at the expected site of base editing following C3 LNP-mediated base editing in primary MPS-I murine fibroblasts at three ABE/sgRNA mass ratios. (F) Sequencing results in primary MPS-I murine neurons treated with C3.MPS LNPs at a range of doses. * p < 0.05, ** p < 0.01, *** p < 0.001 by one-way ANOVA with post-hoc Dunnett’s test compared positive controls described in the text. Outliers were detected using Grubbs’ test and removed from analysis; minimum n = 3 replicates per treatment group; error bars represent SEM.

Article Snippet: LNP formulation Ionizable lipids, prepared as described above, or DLin-MC3-DMA (MedChem Express) were combined in an ethanol phase with cholesterol (Sigma-Aldrich), DOPE (Avanti), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (C14-PEG2000, Avanti) at a molar ratio of 35:46.5:16:2.5 or at molar ratios specified in Fig. S6 .

Techniques: Formulation, Fluorescence, In Vitro, Sequencing

Journal: Nature biomedical engineering

Article Title: Screening for lipid nanoparticles that modulate the immune activity of helper T cells towards enhanced antitumour activity

doi: 10.1038/s41551-023-01131-0

Figure Lengend Snippet: a, Schematic of the screening method and the therapeutic mechanism of mRNA LNP vaccination against a solid tumour. In vitro transfection efficiency was assessed for 1,080 LNP formulations with different helper lipids and component ratios. The top-performing formulations were then tested on BMDCs for transfection and antigen presentation, and in vivo immune responses induced by selected LNPs were assessed. LNPs transfect tissue-resident DCs following s.c. injection, or drain into the neighboring lymph nodes where they transfect APCs including DCs. These APCs translate and process the mRNA into peptides presented on major histocompatibility complex molecules on the cell surface. The lipids also trigger activation pathways that promote co-stimulatory molecule expression and cytokine release. T cells activated by the APCs proliferate and travel to the tumour site to kill cancer cells in an antigen-specific manner. TAP, transporter associated with antigen processing; TCR, T cell receptor; TNF-α, tumour necrosis factor α. b, DC2.4 cells were treated with fLuc mRNA LNPs (1 μg ml−1). The relative luciferase expression after 24 h incubation with fLuc mRNA LNPs is shown in a heat map. c, BMDCs were treated with the 49 top-performing LNPs packaged with mCherry mRNA. The percentage of mCherry+ cells gated on CD11c+ cells after 24 h incubation with mRNA LNPs is shown. LNP formulation details are shown in pie charts with DLin-MC3-DMA in red, cholesterol in green, DMG-PEG2000 in yellow and helper lipids in blue. The top seven formulations, indicated by red arrows, were selected for further study. d–f, Antigen presentation (d), with maturation levels of BMDCs (e,f) were analysed by flow cytometry after 24 h incubation with the seven mOVA-loaded LNPs, PBS, free OVA, or LPS and SIINFEKL peptide. The percentages of SIINFEKL-H-2Kb+ cells (d), additionally positive for CD86 (e) or CD40 (f) gated on CD11c+ cells are shown. g, Representative flow cytometry analysis of SIINFEFL-H-2Kb and CD40 expression on BMDCs treated with the three top-performing LNPs. h, Secretion levels of IFN-γ, TNF-α and IL-6 within the supernatant of BMDCs after 24 h incubation with the three mOVA-loaded LNPs were measured by ELISA and are shown in a radar chart. Data represent the mean ± s.e.m. from a representative experiment (n = 3 (b–g), n = 4 (h) biologically independent samples) of two independent experiments. Data were analysed using one-way ANOVA and Dunnett’s multiple comparisons test against the PBS control group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, not significant.

Article Snippet: Materials DLin-MC3-DMA was purchased from MedKoo Biosciences.

Techniques: In Vitro, Transfection, In Vivo, Injection, Activation Assay, Expressing, Luciferase, Incubation, Formulation, Flow Cytometry, Enzyme-linked Immunosorbent Assay