(A) Polymerization of sarcosine NCA with dioctadecyl/tetradecylamine as initiator followed by end-capping with azidobutyric acid pentafluorophenylester. BA_C18-pSar₃₀-Azide was characterized by (B) ¹H NMR in DMSO‑d₆, and (C) ¹H DOSY NMR in DMSO‑d₆. (D) Analytical gel permeation chromatography in HFIP of (D1) BA_C18-pSar_n-Azide (n = 30, 65) and (D2) BA_C14-pSar_n-Azide (n = 36, 77).

(A-C) Schematic of liposomes comprising of (A) DOTAP, DOPE with shielding lipid, either (B) pSar-lipid or DSPE-PEG2k at different molar fraction. (D) Hydrodynamic diameter and ζ potential of liposomes in HEPES buffer (10 mM, pH 7.4) were determined by DLS (n = 3).

Lipoplexes containing GFP mRNA and PE-LR labeled liposomes were incubated with (A) Jurkat T cells and (B) Hela cells for 24 h to examine transfection efficiency. Lipoplexes were prepared by incubating equal volume of mRNA and liposomes at N/P 10. At the end of incubation, cells were harvested and quantified by flow cytometry, as demonstrated in (A1, A2) of Jurkat T cells and (B1, B2) Hela cells. Representative images of (A3) Jurkat T cells and (B3) Hela cells, after transected with mRNA lipoplexes composed of 2.5% DSPE-PEG2k or pSar-lipid-polyplexes. All the data was averaged from three independent experiments (n = 9).

Biodistribution of liposomes (1 nL, 30 mM) modified with (B) DSPE-PEG2k, (C) C14-pSar2k, (D) C14-pSar5k, (E) C18-pSar2k and (F) C18-pSar5k after intravenous injection into 2.5 dpf zebrafish embryos. (A) Representative images of whole embryo view at 1 h after administration with 10% PEGylated liposomes. Tg (Kdrl:GFP) zebrafish embryos expressed GFP (green) within all endothelial cells (ECs). Common cardinal vein (CCV) and posterior cardinal vein (PCV) are the two main venous vessels, as green arrows indicate. Different blood vessels were indicated by white arrows: dorsal aorta (DA), caudal hematopoietic tissue (CHT), caudal vein (CV), intersegmental vessel (ISV), dorsal longitudinal anastomotic vessel (DLAV). PE-LR labeled liposomes were shown in magenta. Tissue level views (40× lens, magnification of the white square) displayed the merged image of liposomes (magenta) and ECs (green) and separated images in gray. (B^---F) Representative biodistribution images of (B) 2.5%, 5.0% and 10% PEGylated liposomes (C) C14-pSar2k-, (D) C14-pSar5k-, (E) C18-pSar2k- and (F) C18-pSar5k-coated liposomes in 2.5 dpf zebrafish embryos at 1 hpi and 5 hpi. White arrows show liposome aggregates at 1 hpi, and red triangles show that the liposomes were internalized into macrophages. Scale bar: 200 μm for whole embryo view and 50 μm for tissue level view. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

GFP expression within 2.5 dpf zebrafish embryos at 24 hpi in the dorsal view. 1.5 nL lipoplexes (DOTAP/DOPE/DSPE-Rhodamine B/pSar-lipid or DSPE-PEG2k, 30 mM, N/P 10) containing GFP mRNA (100 ng/μL) were injected into the zebrafish brain. (A-C) GFP (green) expression in wildtype ABTL zebrafish embryos that were injected with (A1-A4) 2.5% C14-pSar2k, C14-pSar5k, C18-pSar2k and DSPE-PEG2k lipoplexes (magenta) and (B1-B4) 5.0% C14-pSar2k, C14-pSar5k, C18-pSar2k and DSPE-PEG2k lipoplexes in dorsal view (scale bar: 200 μm). (C) Quantification of GFP-expressing cells in the brain of zebrafish embryos after intracranial injection (n = 6). (D) GFP (green) expression within 2.5 dpf Tg(mpeg1: RFP) zebrafish embryos (macrophages in magenta) at (D1) 24 hpi, (D2) 48 hpi and (D3) 72 hpi in the dorsal view of brain (scale bar: 200 μm, 100 μm, 50 μm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Biodistribution of lipoplexes composed of fluorescently labeled liposomes (megenta, DOTAP/DOPE/DSPE-Rhodamine /C14-pSar2k at a molar ratio of 48.65/48.65/0.2/2.5, 30 mM) and Atto488 labeled luciferase mRNA (green, 150 pg mRNA, N/P 10) (A) in brain after intraventricular injection and (B) in whole embryo after intravenous injection in 2.5 dpf ABTL zebrafish embryos. Confocal images were taken by 10× lens for whole embryos view (scale bar: 100 μm) and 40× lens (scale bar: 20 μm) at 1 hpi, 2 hpi, 4 hpi and 24 hpi. Co-localized liposomes and mRNA were shown in red square. Free liposomes were marked by orange arrows in the zoomed images, respectively. F: forebrain; M: midbrain; H: hindbrain. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)