Journal: Biomaterials science

Article Title: In vivo assessment of triazine lipid nanoparticles as transfection agents for plasmid DNA.

doi: 10.1039/d2bm01289h

Figure Lengend Snippet: Fig. 3 PEG550, DOPE, and TZ3 yield improved transfection with LNP. (A and B) HEK293T cells were transfected with 200 ng GFP plasmid per well using LNPs and analyzed three days later for GFP expression by flow cytometry. (A) LNPs formulated with 50% TZ3, 10% DPSC, 39% cholesterol and 1% DSPE-PEG(550–2000), or 40% cholesterol and no PEG. (B) LNPs formulated with 50% DOTAP (Do) or TZ3, 10% DSPC or DOPE, 39% cholesterol and 1% DSPE-PEG550. Pooled data from three independent experiments are shown; n = 3 transfected wells per group per experiment. (C–F) Equal numbers of male and female BALB/c mice (6 total mice in untreated group (NT), 8 total mice in treated groups) were administered 1 × 109 genome copies of AAV8-GFP or 10 µg of GFP plasmid in either LNPs made with 50% TZ3, 10% DOPE, 39% cholesterol and 1% DSPE-PEG550 or LPs made with 50% TZ3 and 50% DOPE. One week post administration, hepatocytes were evaluated for percent GFP positive cells (C) or mean fluorescence intensity (MFI; D). Percent weight change (E) and serum ALT (F) were evaluated at 72 hours post-administration. Bars indicate mean transfection efficiency; dots represent individual transfection wells (A) or mice (C–F). Data were compared with one-way ANOVA and Dunnett’s post-hoc test (A and C–F) or Sidak’s test (B); comparisons shown in (A) are to No PEG and in (C–F) to untreated (NT), only significant comparisons are shown.

Article Snippet: For in vivo evaluation of GFP transfection, mice were administered GFP plasmid (Addgene no. 37825) i.p. using LNPs or LPs at a dose of 10 μg of DNA or AAV8 at a dose of 2 × 109 genome copies per mouse (equating to approximately 200 ng of DNA) and serum was collected 3 days later to evaluate ALT levels as described above.

Techniques: Transfection, Plasmid Preparation, Expressing, Cytometry

Journal: Biomaterials science

Article Title: In vivo assessment of triazine lipid nanoparticles as transfection agents for plasmid DNA.

doi: 10.1039/d2bm01289h

Figure Lengend Snippet: Fig. 6 PEGylation decreases LNP uptake by antigen presenting cells. (A) Bone marrow-derived dendritic cells (DC) or J774 macrophages were incubated for 18 hours with LNPs made with 5% DiD and DSPE-PEG2000, or PEG-free liposomes. The percentage of cells positive for DiD fluorescence by flow cytometry is shown; data represent pooled results from three independent experiments, N = 3 wells per treatment. (B) Bone marrow-derived dendritic cells or J774 macrophages were transfected with 200 ng of GFP DNA delivered with LPs made of 50% TZ3 and 50% DOPE; LNPs made with 50% TZ3, 10% DOPE, 40% chole- sterol; or LNPs made with 50% TZ3, 10% DOPE, 39% cholesterol and 1% DSPE-PEG550. Seventy-two hours after transfection, the cells were ana- lyzed by flow cytometry for GFP expression; data represent pooled results from three independent experiments, N = 3 wells per treatment. Bars indicate mean ± SD. Only statistically significant comparisons are shown. Significance determined by one-sample T-test (A) or one-way ANOVA (B).

Article Snippet: For in vivo evaluation of GFP transfection, mice were administered GFP plasmid (Addgene no. 37825) i.p. using LNPs or LPs at a dose of 10 μg of DNA or AAV8 at a dose of 2 × 109 genome copies per mouse (equating to approximately 200 ng of DNA) and serum was collected 3 days later to evaluate ALT levels as described above.

Techniques: Derivative Assay, Incubation, Liposomes, Cytometry, Transfection, Expressing

Journal: bioRxiv

Article Title: A prelimbic molecular clock of protein synthesis for memory persistence

doi: 10.64898/2026.01.02.697403

Figure Lengend Snippet: a ) Schematic of in vivo fiber photometry showing AAV1.CAG.GCaMP6f injection and optic ferrule implant into the prelimbic (PL) cortex of wildtype mice to monitor neural activity via virally encoded GCaMP6f expression. Scale bars, 500 μm and 50 μm. b) Schematic of longitudinal and nonlongitudinal testing of remote LTM in Pavlovian threat conditioning (PTC) paradigm. Mice received two pairings of an auditory conditioned stimulus (CS) co-terminating with a foot shock (US) during training in Context A, followed by tests of recent and remote long-term memory (LTM) in Context B in longitudinal framework or that of single timepoint remote LTM at 28d post-training (non-longitudinal, NL). c) Representative heatmaps showing GCaMP6f fluorescence (ΔF/F) and freezing response during pre-CS, CS, and post-CS epochs (30s each) during training. Peri-event time histograms (PETH), mean+/-SEM, show that the first CS–US pairing elicited large PL calcium transients in response to the US, which decreased significantly during the second pairing, indicating experience-dependent attenuation of neural responses. d) Area-under-the-curve (AUC) analysis revealed significant differences between trials specifically during the post-CS/US period. Trial x CS Epoch: F (2, 12) = 4.335; CS Epoch: F (2, 12) = 8.274; n=5 mice/group. e) Freezing responses were significantly elevated during the second CS, reflecting behavioral learning. F (1.548, 15.48) = 8.549; n=11 animals/group. f) Representative heatmaps showing freezing response during pre-CS, CS, and post-CS epochs (30s each) during remote memory retrieval in the non-longitudinal (NL) group. PETH (mean+/-SEM) for ΔF/F z-scored traces demonstrate robust PL calcium activity during CS retrieval in the NL remote condition. g, h) Representative heatmaps showing freezing response during pre-CS, CS, and post-CS epochs (30s each) during recent and remote memory retrieval in the longitudinal (NL) group. PETH (mean+/-SEM) for ΔF/F traces demonstrate robust PL calcium activity during recent retrieval and retrieval-dependent modulation of remote memory. i) AUC analysis revealed significantly reduced CS-evoked PL activity during remote retrieval relative to recent memory in longitudinal conditions. CS Epoch X LTM: F (4, 162) = 3.841, CS Epoch: F (2, 162) = 18.43; n=5-8 mice/group. j) Freezing responses were significantly diminished at the remote timepoint relative to recent retrieval in longitudinal testing. F (2, 48) = 6.073; n=15-18 mice/group. k) Correlation analysis between CS-evoked calcium transients and freezing behavior during retrieval. r 2 =0.3860. Statistical tests: d, i) Two-way ANOVA with Bonferroni post-hoc test; e) RM One-way ANOVA with Bonferroni post-hoc test; j) One-way ANOVA with Bonferroni post-hoc test; k) Pearson correlation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns not significant.

Article Snippet: The following viral vectors were used: AAV9.CamKII0.4.Cre.SV40 (Addgene, Catalog # 105558, 1.00 X 10^13 GC/ml), AAV8.hSyn.DIO.EGFP (Addgene, Catalog #50457, 1.00 X 10^13 GC/ml), AAV9.EF1a.DIO.GFP-L10 (Addgene Catalog #98747, 1.00 X 10^13 GC/ml), AAV1.CAG.GCaMP6f (Addgene, Catalog #100836, 6.80 X 10^13 GC/ml), and AAV1.EF1a-DIO.tTA (Addgene Catalog #99121, 1.00 × 10^13 GC/ml).

Techniques: In Vivo, Injection, Activity Assay, Expressing, Fluorescence