Gene delivery through the use of a hyaluronate-associated intracellularly degradable crosslinked polyethyleneimine

Abstract

For a non-viral gene delivery system to be clinically effective, it should be non-toxic, compatible with biological components, and highly efficient in gene transfection. With this goal in mind, we investigated the gene delivery efficiency of a ternary complex consisting of DNA, an intracellularly degradable polycation, and sodium hyaluronate (DPH complex). Here, we report that the DPH ternary complex achieved significantly higher transfection efficiency than other polymer systems, especially in the presence of serum. The high transfection efficiency and serum tolerance of DPH are attributed to a unique interplay between CLPEI and HA, which leads to (i) the improved stability of DNA in the extracellular environment and at the early stage of intracellular trafficking and (ii) timely dissociation of the DNA-polymer complex. This study reinforces findings of earlier studies that emphasized each step as a bottleneck for efficient gene delivery; yet, it is the first to show that it is possible to overcome these obstacles simultaneously by taking advantage of two distinctive approaches.

Figure 1

(A) Schematic of DNA-CLPEI-HA (DPH) ternary complex. (B) Transfection efficiency of DNA-polymer complexes in NIH/3T3 fibroblasts. (C) Transfection efficiency of DPH complexes with different HA/DNA ratios in M109 cells. DL, DB, and DP: binary complexes of pEGFP and LPEI (linear PEI 25kDa), BPEI (branched PEI 25kDa), or CLPEI; DLH, DBH, and DPH: ternary complexes of pEGFP, corresponding polymers (LPEI, BPEI, and CLPEI), and HA; Lipo2K: Lipofectamine^™2000. Results are presented as means and standard deviations of three experiments.

Figure 2

(A, B) Cytotoxicity of Lipo2K, BPEI, CLPEI, and a mixture of CLPEI and HA on (A) NIH/3T3 cells and (B) M109 cells. Results are presented as means and standard deviations of three experiments. Arrows indicate polymer concentrations used in transfection experiments: Lipo2K 4.17 μg/mL, BPEI 4.41 μg/mL, and CLPEI and CLPEI/HA 8.82 μg/mL. (C) % Fraction of Cy5-positive cells at 3, 6, and 24 hours after incubation. Results are presented as means and standard deviations of three experiments. (D) A representative flow cytometry histogram showing the cellular uptake of DNA-polymer complexes in 6 hours. (E) Confocal microscope images of subcellular localization of DNA-polymer complexes in NIH/3T3 fibroblasts after 6 hours of transfection. Green fluorescence shows LysoTracker (lysosomes); Red Cy3-labeled DNA; Blue DRAQ5-stained nuclei. The bottom row is the merged view of the above three images. Scale bar = 10 μm.

Figure 3

Agarose gel electrophoresis of DNA-polymer complexes incubated in (A) DNase and/or heparin and (B) 50% CS and/or heparin. The bar graphs below the gel images compare the band intensities obtained by densitometry. Results are presented as means and standard deviations of three experiments. The bands in the dotted boxes were excluded from the analysis because they were artifacts from CS. The bottom tables show the pairs of (A) Smear1 or (B) Smear (indicated on the gel images) band intensities compared by the paired t-test and the p-values.

Figure 4

(A, B) Effect of the intracellular GSH level on transfection efficiencies of DNA-polymer complexes. NIH/3T3 fibroblasts were treated with (A) GSH-MME or (B) BSO to increase or decrease the intracellular GSH, respectively, in the presence of 10% serum. Results are presented as means and standard deviations of three experiments. (C) Representative FRET spectra of DNA-polymer complexes acquired at 3 hours and 24 hours after internalization of the complexes. pBR322 plasmid was dual-labeled with Cy3 and Cy5. (D) Ratio of FRET (Cy5) signal to Cy3 signal (I_660-nm/I_565-nm), which represents tightness of the complex, at 3- and 24-hour post-transfection. All results are presented as means and standard deviations of three experiments and compared by the t-test (*: p < 0.05; **: p < 0.01).