Advanced search
Publication Cover
International Journal of Nanomedicine Volume 7, 2012 - Issue
Submit an article Journal homepage
67
Views
4
CrossRef citations to date
0
Altmetric
Original Research

Degradable copolymer based on amphiphilic N-octyl-N-quatenary chitosan and low-molecular weight polyethylenimine for gene delivery

Chengchu Liu1 Department of Biopharmaceutics, College of Food Science and Technology, Shanghai Ocean University, Shanghai, China;2 Shanghai Engineering Research Center of Aquatic-Product Processing and Preservation, Shanghai, China
,
Qing Zhu1 Department of Biopharmaceutics, College of Food Science and Technology, Shanghai Ocean University, Shanghai, China
,
Wenhui Wu1 Department of Biopharmaceutics, College of Food Science and Technology, Shanghai Ocean University, Shanghai, China
,
Xiaolin Xu1 Department of Biopharmaceutics, College of Food Science and Technology, Shanghai Ocean University, Shanghai, China
,
Xiaoyu Wang3 Department of Pharmaceutics, Changhai Hospital, Second Military Medical University, Shanghai China
,
Shen Gao3 Department of Pharmaceutics, Changhai Hospital, Second Military Medical University, Shanghai China
&
Kehai Liu1 Department of Biopharmaceutics, College of Food Science and Technology, Shanghai Ocean University, Shanghai, ChinaCorrespondence[email protected]
 show all
Pages 5339-5350 | Published online: 08 Oct 2012

Figures & data

Figure 1 Synthetic scheme of OTMCS–PEI. First the N-octyl-N-quatenary chitosan (OTMCS) was synthesized. Then the OTMCS was crosslinked with PEI by N-hydroxysuccinimide.

Abbreviation: OTMCS–PEI, amphiphilic chitosan cross-linked with low-molecular weight polyethylenimine.

Figure 1 Synthetic scheme of OTMCS–PEI. First the N-octyl-N-quatenary chitosan (OTMCS) was synthesized. Then the OTMCS was crosslinked with PEI by N-hydroxysuccinimide.Abbreviation: OTMCS–PEI, amphiphilic chitosan cross-linked with low-molecular weight polyethylenimine.

Figure 2 Representative 1H NMR spectra of (A) chitosan, (B) OTMCS, (C) PEI, and (D) OTMCS–PEI, in D2O at room temperature.

Abbreviations: OTMCS, amphiphilic chitosan; PEI, low-molecular weight polyethylenimine; OTMCS–PEI, amphiphilic chitosan cross-linked with low-molecular weight polyethylenimine; D2O, deuterium oxide.

Figure 2 Representative 1H NMR spectra of (A) chitosan, (B) OTMCS, (C) PEI, and (D) OTMCS–PEI, in D2O at room temperature.Abbreviations: OTMCS, amphiphilic chitosan; PEI, low-molecular weight polyethylenimine; OTMCS–PEI, amphiphilic chitosan cross-linked with low-molecular weight polyethylenimine; D2O, deuterium oxide.

Figure 3 Determination of the buffering capacity of PEI 25 KDa and the OTMCS–PEI polymer by acid-base titration.

Note: Solution containing the polymer (0.2 mg/mL) was adjusted to pH 10.0, and then titrated with HCl from 10.0 to 3.0.

Abbreviation: OTMCS–PEI, amphiphilic chitosan cross-linked with low-molecular weight polyethylenimine.

Figure 3 Determination of the buffering capacity of PEI 25 KDa and the OTMCS–PEI polymer by acid-base titration.Note: Solution containing the polymer (0.2 mg/mL) was adjusted to pH 10.0, and then titrated with HCl from 10.0 to 3.0.Abbreviation: OTMCS–PEI, amphiphilic chitosan cross-linked with low-molecular weight polyethylenimine.

Figure 4 Degradation of OTMCS–PEI.

Notes: The polymer was dissolved in 0.1 M PBS (pH = 7.4) and incubated at 37°C and 100 rpm. Determination of molecular weight (MW) was measured by gel permeation chromatography with multiangle laser light scattering (GPC-MALLS) (n = 3).

Abbreviations: OTMCS–PEI, amphiphilic chitosan cross-linked with low-molecular weight polyethylenimine; PBS, phosphate-buffered solution.

Figure 4 Degradation of OTMCS–PEI.Notes: The polymer was dissolved in 0.1 M PBS (pH = 7.4) and incubated at 37°C and 100 rpm. Determination of molecular weight (MW) was measured by gel permeation chromatography with multiangle laser light scattering (GPC-MALLS) (n = 3).Abbreviations: OTMCS–PEI, amphiphilic chitosan cross-linked with low-molecular weight polyethylenimine; PBS, phosphate-buffered solution.

Figure 5 (A) Particle sizes (nm) of the OTMCS–PEI/DNA complex at w/w ratios of 5, 10, 20, and 40. (B) Zeta potentials (mV) of the OTMCS–PEI/DNA complex at w/w ratios of 5, 10, 20, and 40. The data were expressed as mean values (±standard deviations, n = 3). (C) Transmission electron micrograph of OTMCS–PEI/DNA nanoparticles.

Abbreviation: OTMCS–PEI, amphiphilic chitosan cross-linked with low-molecular weight polyethylenimine.

Figure 5 (A) Particle sizes (nm) of the OTMCS–PEI/DNA complex at w/w ratios of 5, 10, 20, and 40. (B) Zeta potentials (mV) of the OTMCS–PEI/DNA complex at w/w ratios of 5, 10, 20, and 40. The data were expressed as mean values (±standard deviations, n = 3). (C) Transmission electron micrograph of OTMCS–PEI/DNA nanoparticles.Abbreviation: OTMCS–PEI, amphiphilic chitosan cross-linked with low-molecular weight polyethylenimine.

Figure 6 (A) Agarose gel electrophoresis of the OTMCS–PEI/DNA complex at various w/w ratios. Protection of OTMCS–PEI on plasmid DNA. (B) Protection of plasmid DNA from degradation by DNase I at different concentrations of 0, 0.375, 0.75, 1, 1.5, 2.25, and 3 U DNase I/μg DNA. (C) Protection of plasmid DNA from dissociation by serum at varying concentrations of 10%, 25%, and 50%. The lanes 10%, 25%, and 50% without “+” refer to the presence of naked DNA with 10%, 25%, and 50% serum; the lanes 10%, 25%, and 50% with “+” refer to the presence of OTMCS–PEI/DNA complex at w/w ratio 20 besides 10%, 25%, and 50% serum. (D) Protection of plasmid DNA from dissociation by sodium heparin at varying concentrations of 700, 800, 900, 1000, 1100, and 1200 μg/mL.

Abbreviation: OTMCS–PEI, amphiphilic chitosan cross-linked with low-molecular weight polyethylenimine.

Figure 6 (A) Agarose gel electrophoresis of the OTMCS–PEI/DNA complex at various w/w ratios. Protection of OTMCS–PEI on plasmid DNA. (B) Protection of plasmid DNA from degradation by DNase I at different concentrations of 0, 0.375, 0.75, 1, 1.5, 2.25, and 3 U DNase I/μg DNA. (C) Protection of plasmid DNA from dissociation by serum at varying concentrations of 10%, 25%, and 50%. The lanes 10%, 25%, and 50% without “+” refer to the presence of naked DNA with 10%, 25%, and 50% serum; the lanes 10%, 25%, and 50% with “+” refer to the presence of OTMCS–PEI/DNA complex at w/w ratio 20 besides 10%, 25%, and 50% serum. (D) Protection of plasmid DNA from dissociation by sodium heparin at varying concentrations of 700, 800, 900, 1000, 1100, and 1200 μg/mL.Abbreviation: OTMCS–PEI, amphiphilic chitosan cross-linked with low-molecular weight polyethylenimine.

Figure 7 Cytotoxicity of OTMCS–PEI at various concentrations in HeLa cell lines using the MTT assay.

Notes: The data were expressed as mean values (±standard deviations, n = 3); **P < 0.01.

Abbreviations: OTMCS–PEI, amphiphilic chitosan cross-linked with low-molecular weight polyethylenimine; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide.

Figure 7 Cytotoxicity of OTMCS–PEI at various concentrations in HeLa cell lines using the MTT assay.Notes: The data were expressed as mean values (±standard deviations, n = 3); **P < 0.01.Abbreviations: OTMCS–PEI, amphiphilic chitosan cross-linked with low-molecular weight polyethylenimine; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide.

Figure 8 GFP gene transfection in HeLa cells by OTMCS–PEI at w/w ratios of 5, 10, 20, and 30. (A) Percentage of GFP gene transfection in HeLa cells by flow cytometry analysis.* (B) Representative fluorescence images for s transfection in HeLa cells using OTMCS–PEI at various w/w ratios.

Note: *The mean ± standard deviation, n = 6.

Abbreviations: GFP, green fluorescent protein; OTMCS–PEI, amphiphilic chitosan cross-linked with low-molecular weight polyethylenimin.

Figure 8 GFP gene transfection in HeLa cells by OTMCS–PEI at w/w ratios of 5, 10, 20, and 30. (A) Percentage of GFP gene transfection in HeLa cells by flow cytometry analysis.* (B) Representative fluorescence images for s transfection in HeLa cells using OTMCS–PEI at various w/w ratios.Note: *The mean ± standard deviation, n = 6.Abbreviations: GFP, green fluorescent protein; OTMCS–PEI, amphiphilic chitosan cross-linked with low-molecular weight polyethylenimin.

Figure 9 (A) Transfection efficiency of OTMCS–PEI/DNA complex in HeLa cell line at w/w ratios of 5, 10, 20, and 30.* (B) Transfection efficiency of pGL3-Control as a reporter gene in mice.

Notes: BALB/c athymic mice were inoculated with B16 cells. Luciferase gene expression was determined after administration of OTMCS–PEI/DNA complex (w/w = 10) OTMCS–PEI/DNA complex (w/w = 30), and PEI 25 KDa/DNA complex (N/P = 5);† *each data point represents the mean ± standard deviation (n = 6); **P < 0.01; †results were expressed in RLU/mg protein.

Abbreviations: OTMCS–PEI, amphiphilic chitosan cross-linked with low-molecular weight polyethylenimine; RLU, relative light units.

Figure 9 (A) Transfection efficiency of OTMCS–PEI/DNA complex in HeLa cell line at w/w ratios of 5, 10, 20, and 30.* (B) Transfection efficiency of pGL3-Control as a reporter gene in mice.Notes: BALB/c athymic mice were inoculated with B16 cells. Luciferase gene expression was determined after administration of OTMCS–PEI/DNA complex (w/w = 10) OTMCS–PEI/DNA complex (w/w = 30), and PEI 25 KDa/DNA complex (N/P = 5);† *each data point represents the mean ± standard deviation (n = 6); **P < 0.01; †results were expressed in RLU/mg protein.Abbreviations: OTMCS–PEI, amphiphilic chitosan cross-linked with low-molecular weight polyethylenimine; RLU, relative light units.

Related research

People also read lists articles that other readers of this article have read.

Recommended articles lists articles that we recommend and is powered by our AI driven recommendation engine.

Cited by lists all citing articles based on Crossref citations.
Articles with the Crossref icon will open in a new tab.