Cell-Penetrating Peptide-Based Triple Nanocomplex Enables Efficient Nuclear Gene Delivery in Chlamydomonas reinhardtii

Abstract

Microalgae are a promising solution for mitigating climate change due to their ability to capture greenhouse gases and produce renewable materials. However, their effective application is often hindered by barriers that necessitate advances in genetic engineering to improve photosynthesis and productivity. One major obstacle is the microalgal cell wall, which complicates the delivery of genetic material into these organisms. To address these challenges, we developed a novel triple nanocomplex system integrating cell-penetrating peptides (CPPs), nuclear localization signal (NLS) peptides, and plasmid DNA. This system allows simple preparation while achieving efficient nuclear translocation of plasmid DNA. We evaluated two CPPs, pVEC-ORI and pVEC-R6A, for their efficacy in facilitating intracellular transfer of DNA into wild-type Chlamydomonas reinhardtii cells. Notably, pVEC-R6A demonstrated a 6.88-fold increase in efficiency compared to pVEC-ORI, despite the presence of thick cell walls. The optimal CPP:DNA ratio for stable nanocomplex formation was determined to be 5:1 for pVEC-ORI and 10:1 for pVEC-R6A. By incorporating the simian virus 40 (SV40) NLS into CPP/DNA nanocomplexes, we successfully directed the localization of plasmid DNA into the nucleus. Our findings indicate that this simple and efficient DNA delivery system has significant potential as a tool to advance microalgal synthetic biology.

Keywords: SV40; cell‐penetrating peptide; gene delivery; microalgae; nanocomplex; nuclear localization signal; pVEC.

Figure 1

Cell penetration and translocation of cell‐penetrating peptides (CPPs) in microalgae. (a) Schematic illustration of CPP uptake by microalgal cells. (b) Confocal microscopy images showing the translocation of fluorescein isothiocyanate (FITC)‐labeled pVEC‐ORI and pVEC‐R6A peptides in Chlamydomonas reinhardtii. Cells were incubated with 25 μM of pVEC‐ORI and pVEC‐R6A for 1 h at 25°C. After incubation, cells were harvested by centrifugation and washed with nuclease‐free water before analysis by confocal laser scanning microscopy. Scale bar, 5 μM. (c) Fluorescence intensity of FITC in microalgal cells 1 h after treatment with 25 μM FITC‐labeled pVEC‐ORI and pVEC‐R6A (n = 10). Asterisks in the box plots indicate significant differences in mean values between two treatments, analyzed using Student's t‐test. Statistical significance is indicated by ***p < 0.001.

Figure 2

Formation and characterization of CPP/DNA nanocomplex. (a) Agarose gel electrophoresis of CPP/DNA nanocomplexes. 0.5 μg of pChlamy4 plasmid DNA (3640 bp) was mixed with pVEC‐ORI and pVEC‐R6A at varying CPP:DNA ratios (0.1:1, 1:1, 2.5:1, 5:1, 10:1, 15:1, and 20:1). Positive control (PC) consisted of naked plasmids. (b) Size and (c) zeta potential of CPP/DNA nanocomplexes measured at CPP:DNA ratios of 5:1 for pVEC‐ORI and 10:1 for pVEC‐R6A using a Zetasizer. Statistical significance is indicated by ***p < 0.001 (Student's t‐test).

Figure 3

Translocation of CPP/DNA nanocomplexes in microalgal cells. (a) Proposed mechanism of cellular uptake of FITC‐labeled CPP/DNA nanocomplexes and CPP/Cy3‐labeled DNA nanocomplexes in microalgae. (b) Cellular internalization of FITC‐labeled pVEC‐ORI and pVEC‐R6A in complex with plasmid DNA at CPP:DNA ratios of 5:1 and 10:1, respectively. (c) Cellular internalization of Cy3‐labeled plasmid DNA in free form or in complex with CPP, analyzed via confocal laser scanning microscopy. Fluorescence intensity was measured at 516 nm for FITC (d) and 560–580 nm for Cy3 (e). Statistical significance is indicated by **p < 0.01 and ***p < 0.001 (Student's t‐test).

Figure 4

Formation and characterization of triple nanocomplex. Confirmation of triple nanocomplex formation between (a) pVEC‐ORI/DNA/SV40 and (b) pVEC‐R6A/DNA/SV40. The CPP:DNA ratios used were consistent with those previously tested for the formation of CPP/DNA nanocomplexes. SV40 peptide was added at a concentration of 50 μM. Positive control (PC) consisted of naked plasmids. (c) Size and (d) zeta potential of the triple nanocomplexes were measured at CPP:DNA ratios of 2.5:1 for pVEC‐ORI and 5:1 for pVEC‐R6A using a Zetasizer.

Figure 5

Nuclear localization of CPP/DNA/SV40 triple nanocomplexes. (a) Schematic illustration of the triple nanocomplex formation by pVEC‐R6A, Cy3 labeled‐DNA, and SV40 peptide, and the proposed cellular uptake mechanism. (b) Cellular internalization of Cy3‐labeled pChlamy4 in 4′,6‐diamidino‐2‐phenylindole (DAPI)‐stained microalgal cells after treatment with the triple nanocomplex. White arrows indicate the trajectories of Cy3 and DAPI fluorescence in (c) and (d).