Co-assembled Ca2+ Alginate-Sulfate Nanoparticles for Intracellular Plasmid DNA Delivery

Abstract

Successful gene therapy requires the development of suitable carriers for the selective and efficient delivery of genes to specific target cells, with minimal toxicity. In this work, we present a non-viral vector for gene delivery composed of biocompatible materials, CaCl₂, plasmid DNA and the semi-synthetic anionic biopolymer alginate sulfate (AlgS), which spontaneously co-assembled to form nanoparticles (NPs). The NPs were characterized with a slightly anionic surface charge (Zeta potential [ζ] = -14 mV), an average size of 270 nm, and their suspension was stable for several days with no aggregation. X-ray photoelectron spectroscopy (XPS) validated their ternary composition, and it elucidated the molecular interactions among Ca²⁺, the plasmid DNA, and the AlgS. Efficient cellular uptake (>80%), associated with potent GFP gene expression (22%-35%), was observed across multiple cell types: primary rat neonatal cardiac fibroblasts, human breast cancer cell line, and human hepatocellular carcinoma cells. The uptake mechanism of the NPs was studied using imaging flow cytometry and shown to be via active, clathrin-mediated endocytosis, as chemical inhibition of this pathway significantly reduced EGFP expression. The NPs were cytocompatible and did not activate the T lymphocytes in human peripheral blood mononuclear cells. Proof of concept for the efficacy of these NPs as a carrier in cancer gene therapy was demonstrated for Diphtheria Toxin Fragment A (DT-A), resulting in abrogation of protein synthesis and cell death in the human breast cancer cell line. Collectively, our results show that the developed AlgS-Ca²⁺-plasmid DNA (pDNA) NPs may be used as an effective non-viral carrier for pDNA.

Keywords: alginate sulfate; calcium; gene therapy; plasmid DNA; polymeric nanoparticles.

Graphical abstract

Figure 1

High-Resolution TEM Images of AlgS-Ca²⁺-pDNA NPs (A and B) Dry-TEM micrographs of NPs (2.5 μg/mL AlgS, 25 mM Ca²⁺, and 15 ng/μL pDNA) with gold-labeled AlgS. (C) Cryo-TEM micrographs of complexes (250 mM Ca²⁺ and 150 ng/μL pDNA). (D) Cryo-TEM micrographs of NPs (25 μg/mL AlgS, 250 mM Ca²⁺, and 150 ng/μL pDNA). Scale bars, 500 nm (A) and 100 nm (B–D).

Figure 2

Cytocompatibility of NPs Made with Different Ca²⁺ Concentrations (A) 24 h, (B) 48 h, and (C) 72 h post-exposure to NPs (mean ± SD; *p < 0.05,**p < 0.01,***p < 0.005, Tukey’s multiple comparison test compared to control; n = 3).

Figure 3

Uptake of NPs with Different Ca²⁺ Concentrations by Various Cells and Analysis of AlgS-Ca²⁺-pDNA Co-localization after Cellular Uptake (A and B) ImageStream results showing bright-field (BF) and FITC-pDNA in green for NPs with 2.5 mM Ca²⁺ uptaken by (A) CFs and (B) MDA-MB-231. (C–E) Percent transfected cells counted using ImageStreamX (mean ± SD;*p < 0.05, Sidak multiple comparison test compared to control; n = 3) for (C) MDA-MB-231, (D) CFs, and (E) HepG2. Imaging flow cytometry analysis of AlgS-Ca²⁺-pDNA uptake in MDA-MB-231 cells, 3 h after NP addition in unfixed live cells, is shown. (F) Representative cell images captured by Amnis ImageStreamX flow cytometer. (G) From total cell population, cells that are double positive or negative for fluorescent signals of AlgS-Cy5 nm and pDNA-FITC. (H) Histogram of Bright Detail Similarity (BDS) score between pDNA (FITC) and AlgS (Cy5) images, showing the cell population exhibiting co-localization of pDNA (FITC) and AlgS (Cy5). A score of >1.0 was considered co-localized.

Figure 4

GFP Expression Measured Using Flow Cytometry in Different Cells Transfected with AlgS-Ca²⁺-pDNA NPs NPs were prepared with 5 or 2.5 mM [Ca²⁺] (final concentration in cell medium). Mean + SD; *p < 0.05, Sidak multiple comparison test compared to control for each group.

Figure 5

Uptake Kinetics of AlgS-Ca²⁺-pDNA NPs by MDA-MB-231 Cells Using ImageStreamX and Inhibition of Endocytosis (A) Representative images showing bright-field and fluorescein-pDNA (green) accumulation in cells 3 h post-transfection. (B) Percentage of fluorescent-positive cells over 24 h. (C) Mean fluorescence intensity over 24 h, normalized to the peak value (at 3 h post-transfection). Error bars represent SEM (n = 2). (D) Percentage of EGFP-expressing cells treated with inhibitors normalized to cells transfected with pDNA NPs without exposure of the cells to the chemical inhibitors. Error bars represent SD (n = 3); asterisks denote significant difference with *p < 0.05, **p < 0.01, and ***p < 0.005 by one-way ANOVA (Dunnet test).

Figure 6

The Effect of AlgS-Ca²⁺-pDNA NPs on PBMC Activation Human PBMCs, isolated from five healthy individuals, were incubated for 3 h with NPs composed of a final Ca²⁺ concentration of 5 or 2.5 mM and 3 ng/μL pDNA and 500 nM/mL AlgS (NP concentration of ∼3.2 × 107/mL). Cytokine secretion was detected using ELISA. Anti-CD3 and anti-CD28 Dynabead activation was used as a positive control. (A) IL-2 secretion measured 24 h after activation; (B) IFN-γ secretion measured 48 h after activation; (C) IL-10 secretion measured 48 h after activation; (D) IL-6 secretion measured 24 h after activation; and (E) TNF-α secretion measured 24 h after activation. Data are presented as means ± SEM, and y axis is in logarithmic scale (n = 5; *p < 0.05, **p < 0.01, ****p < 0.0001, one-way ANOVA, Tukey’s multiple comparison test).

Figure 7

Reduced Percentage of GFP-Expressing Cells, 24 h after Being Transfected with Both pEGFP N1 and pDT-A N1, Delivered in Lipofectamine 2000 or AlgS-Ca²⁺-pDNA NPs Results are mean ± SEM, n = 3. Asterisks denote significant difference with ***p < 0.0001.

Figure 8

Proliferation of MDA-MB-231 Cells Transfected with Either pEGFP N1 in AlgS-Ca²⁺ NPs, pDT-A N1 in AlgS-Ca²⁺ NPs, or by pDT-A N1 in Lipofectamine 2000 Control group represents untreated cells. Data are presented as the percentage DNA in treated cells from DNA content of the control group measured on the same day. Results are mean ± SEM, n = 3. Asterisks denote significant difference with *p < 0.05, **p < 0.01, and ***p < 0.005 by one-way ANOVA (Tukey post hoc test).