Enhance transgene responses through improving cellular uptake and intracellular trafficking by bio-inspired non-viral vectors

Abstract

Background: Gene therapy remains a significant challenge due to lots of barriers limiting the genetic manipulation technologies. As for non-viral delivery vectors, they often suffer insufficient performance due to inadequate cellular uptake and gene degradation in endosome or lysosome. The importance of overcoming these conserved intracellular barriers is increasing as the delivery of genetic cargo.

Results: A surface-functionalized non-viral vector involving the biomimetic mannitol moiety is initiated, which can control the cellular uptake and promote the caveolae-mediated pathway and intracellular trafficking, thus avoiding acidic and enzymatic lysosomal degradation of loaded gene internalized by clathrin-mediated pathway. Different degrees of mannitol moiety are anchored onto the surface of the nanoparticles to form bio-inspired non-viral vectors and CaP-MA-40 exhibits remarkably high stability, negligible toxicity, and significantly enhanced transgene expression both in vitro and in vivo.

Conclusions: This strategy highlights a paradigmatic approach to construct vectors that need precise intracellular delivery for innovative applications.

Keywords: Cellular uptake pathway; Intracellular trafficking; Non-viral vectors; Transgene.

Fig. 1

Schematic representation for the cellular uptake and intracellular trafficking of bio-inspired CaP-MA non-viral vectors

Fig. 2

The percentage of positive transfected cells (transfected Cells%, a), mean fluorescent intensity (MFI, b) and green fluorescence protein expression (c) without serum; relative percentage of transfection efficiency (d) and green fluorescence protein expression in the presence of serum (e) observed by fluorescence microscopy (4×). Data are displayed as mean ± SD (n = 3). *P < 0.05, compared with naked DNA, ^#P < 0.05, compared with CaP, ^&P < 0.05, compared with Lipofectamine-2000 (Lip-2000), ^@P < 0.05, compared with serum free group

Fig. 3

Subcellular colocalization of fluorescence-labeled CaP nanoparticles (a, b) and CaP-MA-40 nanoparticles (c, d) with molecular probes of lysosome or caveosome in HEK239T cells observed by Laser Scanning Confocal Microscopy. The colocalization ratio of nanoparticles with lysosomes or caveosome (e). LysoTracker® Deep Red and CT-B were used as specific molecular probes to label lysosome and caveosome, respectively. *P < 0.05, colocalization ratio of CaP with lysosome compared with the counterpart in CaP-MA-40 group, ^#P < 0.05, colocalization rate of CaP with caveosome compared with the counterpart in CaP-MA-40 group

Fig. 4

The influences on the transfection efficiency when treated with specific endocytic inhibitors amiloride (a), chlorpromazine (b) and genistein (c) and cellular uptake of fluorescence labeled CaP (d, e, h) and CaP-MA-40 (f, g, i) nanoparticles after treated with specific endocytic inhibitors in HEK293T cells. Data were represented as the relative transfection efficiency to the untreated cells group. Data are displayed as mean ± SD (n = 3). *P < 0.05, compared with the untreated cells group, ^#P < 0.05, compared with the CaP group

Fig. 5

Evaluation of P-CAV1 expression by western blot experiment. a, b Cells were treated with Mannitol and MA-AL, respectively. c, d Cells were treated with CaP and CaP-MA-40 nanoparticles, respectively. GE + CaP, GE + CaP-MA-40, GE + Mannitol and GE + MA-AL groups were pre-incubated with genistein (GE) and untreated cells as the negative control, mean ± SD, n = 3. *P < 0.05, CaP, CaP-MA-40, Mannitol and MA-AL compared with control, ^#P < 0.05, CaP-MA-40 compared with CaP, ^&P < 0.05, GE + CaP-MA-40 compared with CaP-MA-40, GE + CaP compared with CaP, GE + Mannitol compared with Mannitol, and GE + MA-AL compared with MA-AL

Fig. 6

In vivo transgene efficiency evaluation performed on nude mice. a Fluorescence images of transfected nude mice at different time points after single tail-vein injection of CaP and CaP-MA nanoparticles containing EGFP plasmid. b Fluorescence images of excised organs at 6 h after tail-vein injection. c Frozen section of organs at 6 h after tail-vein injection observed by laser scanning confocal microscopy