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Abstract
Background
Polycation carriers show great foreground in the developing efficient and safe gene delivery; nevertheless, they are cytotoxic and unstable in vivo because of the excess cationic charge. PEGylation improves the biocompatibility and stability of polycation, whereas PEGylation restrains the endosomal escape to some extent.
Materials and methods
To address this issue and promote the transfection in vivo, a pH-sensitive conjugate folate-polyethylene glycol-carboxylated chitosan (shorten as FA-PEG-CCTS) was designed and coated on the surface of PEI/NLS/pDNA (PNDs), forming a versatile gene carrier FA-PEG-CCTS/PEI/NLS/pDNA (FPCPNDs). The novel carrier exhibited a few picturesque characteristics, including (i) neutral surface charge to restrain nonspecific interactions; (ii) folate receptors (FR)-mediated endocytosis to augment cellular uptake; (iii) dual proton sponge effect to realize endosome escape, and (iv) nuclear localization sequences (NLS) to enhance the transfection of pDNA.
Results
FPCPNDs could compress and protect pDNA from degradation. FPCPNDs energetically targeted tumor cells because of their high binding affinity between FA and highly expressed FR on the tumor surface, accordingly enhancing the cellular uptake. In the acidic endosomes, FA-PEG-CCTS segment dissociated from PNDs. Then, PNDs realized endosomal escape through the proton sponge effect of PEI. Furthermore, FPCPNDs showed admirable transfection efficiency with the aid of NLS peptides. What’s more, in vivo studies revealed that FPCPNDs had supreme antitumor activity among the whole preparations.
Conclusion
In vitro and in vivo assays thus demonstrate that FPCPNDs is a hopeful strategy for gene delivery.
Supplementary materials
Figure S1 Synthesis of FA-PEG-CCTS conjugates.
Abbreviations: FA, folate acid; PEG, polyethylene glycol; CCTS, carboxylated chitosan.
Figure S2 1H-NMR analysis of (A) FA, (B) CCTS, (C) mPEG-COOH, and (D) FA-PEG-CCTS.
Abbreviations: FA, folate acid; PEG, polyethylene glycol; CCTS, carboxylated chitosan.
Acid–base titration
The buffering capacity of FA-PEG-CCTS from pH 7.4 to 5.0 was evaluated by acid–base titration. In brief, FA-PEG-CCTS conjugates were dissolved in 0.01 M NaCl (4 mg/mL) and the solution was adjusted to pH 12 with 1 M NaOH. The diluted solution was titrated by the stepwise addition of 0.01 M HCl to obtain the titration profile. The pKa of FA-PEG-CCTS was determined by the primary derivative method and the buffering capacity was defined as the percentage of amino groups from pH 7.4 to 5.0.
Figure S3 Titration curves obtained by titrating aqueous solutions of PEG-CCTS and FA-PEG-CCTS (4 mg/mL) in 0.01 M NaCl (pH 12, adjusted with NaOH) with 0.01 M HCl.
Abbreviations: FA, folate acid; PEG, polyethylene glycol; CCTS, carboxylated chitosan.
Table S1 Buffering capacity and pKa determination by acid–base titration
Western blotting
To evaluate the p53 protein expression level of FPCPNDs, 4T1 cells (1×105 cells/well) were seeded on glass cover slips in 6-well plates and cultured overnight. FPCPNDs were subsequently added to each well and incubated for 4 hrs. After incubation for 48 hrs, the medium was removed and washed with PBS. Afterward, the cells were collected, followed by RIPA lysed on ice for 30 mins. The lysates were centrifuged at 16,000 r/min for 4 mins and the supernatant was determined with an enhanced BCA protein assay kit. Each sample was adjusted to get an identical protein concentration. In brief, each sample was fractionated and concentrated in the spacer gel during SDS-PAGE. Subsequently, the separated protein bands were electrically transferred onto polyvinylidene difluoride (PVDF) membranes. p53 and β-actin primary antibodies and horseradish peroxidase (HRP) conjugated Goat anti-Rabbit IgG were sequentially incubated with the PVDF membranes. The ECL Western blotting substrate was added onto the PVDF membranes and the designated protein bands were visualized by exposed and photographed with MicroChemi 4.2 Gel Imager (DNR, Israel).
Figure S4 The p53 protein expression level of FPCPNDs was examined by Western blotting on 4T1 cells. Cells treated with PBS were set as control.
Abbreviations: FPCPNDs, FA-PEG-CCTS/PEI/NLS/pDNA; PND, PEI/NLS/pDNA; PCPNDs, PEG-CCTS/PEI/NLS/pDNA; FA, folate acid; PEG, polyethylene glycol; CCTS, carboxylated chitosan; PEI, polyethyleneimine.
Figure S5 Changes in size of the FPCPNDs following incubation with RPMI-1640 medium containing 10% FBS for 24 hrs.
Abbreviations: FPCPNDs, FA-PEG-CCTS/PEI/NLS/pDNA; PND, PEI/NLS/pDNA; PCPNDs, PEG-CCTS/PEI/NLS/pDNA; FA, folate acid; PEG, polyethylene glycol; CCTS, carboxylated chitosan; PEI, polyethyleneimine.
Disclosure
The authors report no conflicts of interest in this work.