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. 2020 Feb 18;18(1):34.
doi: 10.1186/s12951-020-0586-8.

Triazine-cored Polymeric Vectors for Antisense Oligonucleotide Delivery in Vitro and in Vivo

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Free PMC article

Triazine-cored Polymeric Vectors for Antisense Oligonucleotide Delivery in Vitro and in Vivo

Mingxing Wang et al. J Nanobiotechnology. .
Free PMC article

Abstract

Background: The polymer-based drug/gene delivery is promising for the treatment of inherent or acquire disease, because of the polymer's structural flexibility, larger capacity for therapeutic agent, low host immunogenicity and less cost. Antisense therapy is an approach to fighting genetic disorders or infections using antisense oligonucleotides (AOs). Unfortunately, the naked AOs showed low therapeutic efficacy in vivo and in clinical trial due to their poor cellular uptake and fast clearance in bloodstream. In this study, a series of triazine-cored amphiphilic polymers (TAPs) were investigated for their potential to enhance delivery of AOs, 2'-O-methyl phosphorothioate RNA (2'-OMePS) and phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo.

Results: TAPs significantly enhanced AO-induced exon-skipping in a GFP reporter-based myoblast and myotube culture system, and observed cytotoxicity of the TAPs were lower than Endoporter, Lipofectamine-2000 or PEI 25K. Application of optimized formulations of TAPs with AO targeted to dystrophin exon 23 demonstrated a significant increase in exon-skipping efficiency in dystrophic mdx mice. The best ones for PMO and 2'-OMePS delivery have reached to 11-, 15-fold compared with the AO only in mdx mice, respectively.

Conclusion: The study of triazine-cored amphiphilic polymers for AO delivery in vitro and in mdx mice indicated that the carrier's performances are related to the molecular size, compositions and hydrophilic-lipophilic balance (HLB) of the polymers, as well as the AO's structure. Improved exon-skipping efficiency of AOs observed in vitro and in mdx mice accompanied with low cytotoxicity demonstrated TAP polymers are potentials as safe and effective delivery carrier for gene/drug delivery.

Keywords: Amphiphilic cationic polymers; Antisense delivery; Exon-skipping; Muscular dystrophy; Triazine.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Cell viability of C2C12E50 myoblasts after treatment with TAPs at 3 doses (5, 10, 25 µg/mL; PEI 25k as control) determined by MTS assay. Cells were seeded in 96-well plate at an initial density of 1 × 104 cells/well in 0.2 mL growth media. The results are presented as the mean ± SD in triplicate
Fig. 2
Fig. 2
Delivery efficiency and toxicity of TAP/2′-OMePS E50 complexes in a C2C12E50 cell line determined by fluorescence microscopy and fluorescence-activated cell sorting (FACS) analysis. a Representative fluorescence images of 2′-OMePS E50-induced exon-skipping in C2C12E50 cell line. (The images were taken 4-day after treatment. Original magnification ×200; scale bar 500 µm). b Statistical date of TE of 2′-OMePS E50 formulated with TAP (Student’s t-test, *p ≤ 0.05 compared with 2′-OMePS E50 only). c Cell viability (Student’s t-test, *p ≤ 0.05 compared with untreated cell as control). In this test, 2 µg 2′-OMePS E50 was formulated with TAPs (5 and 10 µg), and PEI 25 (5 µg), LF-2k (4 µg) formulated as comparison in 0.5 mL 10% FBS-DMEM medium, respectively. The results are presented as the mean ± SD in triplicate
Fig. 3
Fig. 3
Delivery efficiency and toxicity of TAP/PMOE50 complexes in a C2C12E50 cell line determined by fluorescence microscopy and flow cytometry. a Representative fluorescence images of PMOE50-induced exon-skipping in C2C12E50 cell line. (The images were taken 4-day after treatment. Original magnification ×200; scale bar 200 µm). b Statistical date of TE of PMOE50 formulated with TAP (Student’s t-test, *p ≤ 0.05 compared with PMO only). c Cell viability (Student’s t-test, *p ≤ 0.05 compared with untreated cell as control). In this test, 5 µg PMOE50 was formulated with TAPs (10 µg), and PEI 25 (5 µg), Endoporter (5 µg) formulated as comparison in 0.5 mL 10% FBS-DMEM medium, respectively. The results are presented as the mean ± SD in triplicate
Fig. 4
Fig. 4
Green fluorescent protein expression induced by PMOE23 (5 μg) formulated with 1A41B2 dose-dependent in C2C12E23 cells in 0.5 mL of 10% FBS-DMEM after 6-day treatment. Original magnification ×200; scale bar 200 µm
Fig. 5
Fig. 5
Negatively stained transmission electron micrographs of TAPs, PMO only, and TAPs (10 μg) complexed with PMO (5 μg) or 2′-OMePS (2 μg) (scale bar 100 nm)
Fig. 6
Fig. 6
Restoration of dystrophin in tibialis anterior muscles of mdx mice (aged 4–5 weeks) 2 weeks after intramuscular injection with 10 μg polymer formulated PMOE23 (2 μg) in 40 μL saline. Muscles treated with PMOE23 only was used as controls. a Dystrophin was detected by immunohistochemistry with rabbit polyclonal antibody P7 against dystrophin. Blue nuclear staining with 4,6-diamidino-2-phenylindole (original magnification ×200; scale bar 200 µm). b The percentage of dystrophin-positive fibers (mean ± SD, n = 5, Two-tailed t-test, *p ≤ 0.05 compared with PMO). c Detection of exon 23 skipping by RT-PCR. Total RNA of 100 ng from each sample was used for amplification of dystrophin mRNA from exon 20 to exon 26. The upper bands (indicated by E22 + E23 + E24) correspond to the normal mRNA, and the lower bands (indicated by E22 + E24) correspond to the mRNA with exon E23 skipped
Fig. 7
Fig. 7
Restoration of dystrophin in tibialis anterior muscles of mdx mice (aged 4–5 weeks) 2 weeks after intramuscular injection with 20 μg polymer formulated 2′-OMePS (5 μg) in 40 μL saline. Muscles treated with 2′-OMePS only was used as controls. a Dystrophin was detected by immunohistochemistry with rabbit polyclonal antibody P7 against dystrophin. Blue nuclear staining with 4, 6-diamidino-2-phenylindole (original magnification ×200; scale bar 200 μm). b The percentage of dystrophin-positive fibers (mean ± SD, n = 5, Two-tailed t-test, *p ≤ 0.05 compared with 2′-OMePS)
Fig. 8
Fig. 8
Dystrophin expression in different muscles and serum study of mdx mice (aged 4–5 weeks) 2 weeks after systemic administration of PMO with TAPs. Each mouse was injected with 1 mg PMOE23 with and without TAP (0.5 mg). a Immunohistochemistry with antibody P7 for the detection of dystrophin (original magnification ×100; scale bar 200 µm). b Percentage of dystrophin-positive fibers in different muscle tissues (mean ± SD, n = 5, Two-tailed t-test, *p ≤ 0.05 compared with 1 mg PMO)

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