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TAT-LHRH conjugated low molecular weight chitosan as a gene carrier specific for hepatocellular carcinoma cells.

Liu L, Dong X, Zhu D, Song L, Zhang H, Leng XG - Int J Nanomedicine (2014)

Bottom Line: To develop a chitosan-based nonviral gene carrier capable of delivering genes specifically into hepatoma cells, a bifunctional peptide composed of the TAT (transactivator of transcription) peptide and luteinizing hormone-releasing hormone (LHRH) was conjugated with low molecular weight chitosan, resulting in a TAT-LHRH-chitosan conjugate (TLC).In vitro targeting specificity and transfection efficiency were analyzed with a GE IN Cell Analyzer 2000 High-Content Cellular Analysis System.The results demonstrated that TLC had stronger DNA condensing power than unmodified chitosan, and that TLCDNPs were of roughly round shape with average diameter of 70-85 nm and zeta potential of +30 mV and were relatively stable in solution.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bioengineering, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin Key Laboratory of Biomedical Materials, Tianjin, People's Republic of China.

ABSTRACT
To develop a chitosan-based nonviral gene carrier capable of delivering genes specifically into hepatoma cells, a bifunctional peptide composed of the TAT (transactivator of transcription) peptide and luteinizing hormone-releasing hormone (LHRH) was conjugated with low molecular weight chitosan, resulting in a TAT-LHRH-chitosan conjugate (TLC). TLC/DNA nanoparticles (TLCDNPs) were characterized by agarose gel retardation, atomic force microscopy, and dynamic light scattering analysis. In vitro targeting specificity and transfection efficiency were analyzed with a GE IN Cell Analyzer 2000 High-Content Cellular Analysis System. The results demonstrated that TLC had stronger DNA condensing power than unmodified chitosan, and that TLCDNPs were of roughly round shape with average diameter of 70-85 nm and zeta potential of +30 mV and were relatively stable in solution. The in vitro study demonstrated TLC was highly selective for hepatoma cells and essentially nontoxic.

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Infrared spectra of (A) chitosan and (B) TAT-LHRH-chitosan.Abbreviations: TAT-LHRH, transactivator of transcription – luteinizing hormone-releasing hormone; AU, arbitrary units.
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f2-ijn-9-2879: Infrared spectra of (A) chitosan and (B) TAT-LHRH-chitosan.Abbreviations: TAT-LHRH, transactivator of transcription – luteinizing hormone-releasing hormone; AU, arbitrary units.

Mentions: As designed in the current study, TLC should be formed through the disulfide bond linkage between the sulfydryl group from the cysteine residue in the peptide and the pyridyldithiol group generated by the reaction of SPDP with the primary amines (-NH2) in the chitosan. Therefore, the number of primary amines should be reduced and the secondary amines would increase in the resultant conjugates. Figure 2 shows the result of infrared spectroscopy analysis, which revealed that the intensity of the primary amine bond at 1,658.07 cm−1 in the resultant TLC decreased while the secondary amine bond at 1,555.56 cm−1 increased as compared with the unmodified chitosan, indicating the successful conjugation of TAT-LHRH peptides with chitosans. As shown in Figure 3, NMR spectroscopic analysis demonstrated the spectrum of H on the benzene ring of tyrosine peak at 6.8 ppm and that of the H on the primary amines of chitosan at 4.8 ppm. It was estimated that the substitution degree of TLC was about 3% according to the area ratio above the peak. Data from Figure 4 reveal that the isoelectric point of TLC was 11.3, which was significantly higher than that of the unmodified chitosan,33 suggesting that TLC would be more positively charged than the unmodified chitosan.


TAT-LHRH conjugated low molecular weight chitosan as a gene carrier specific for hepatocellular carcinoma cells.

Liu L, Dong X, Zhu D, Song L, Zhang H, Leng XG - Int J Nanomedicine (2014)

Infrared spectra of (A) chitosan and (B) TAT-LHRH-chitosan.Abbreviations: TAT-LHRH, transactivator of transcription – luteinizing hormone-releasing hormone; AU, arbitrary units.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061174&req=5

f2-ijn-9-2879: Infrared spectra of (A) chitosan and (B) TAT-LHRH-chitosan.Abbreviations: TAT-LHRH, transactivator of transcription – luteinizing hormone-releasing hormone; AU, arbitrary units.
Mentions: As designed in the current study, TLC should be formed through the disulfide bond linkage between the sulfydryl group from the cysteine residue in the peptide and the pyridyldithiol group generated by the reaction of SPDP with the primary amines (-NH2) in the chitosan. Therefore, the number of primary amines should be reduced and the secondary amines would increase in the resultant conjugates. Figure 2 shows the result of infrared spectroscopy analysis, which revealed that the intensity of the primary amine bond at 1,658.07 cm−1 in the resultant TLC decreased while the secondary amine bond at 1,555.56 cm−1 increased as compared with the unmodified chitosan, indicating the successful conjugation of TAT-LHRH peptides with chitosans. As shown in Figure 3, NMR spectroscopic analysis demonstrated the spectrum of H on the benzene ring of tyrosine peak at 6.8 ppm and that of the H on the primary amines of chitosan at 4.8 ppm. It was estimated that the substitution degree of TLC was about 3% according to the area ratio above the peak. Data from Figure 4 reveal that the isoelectric point of TLC was 11.3, which was significantly higher than that of the unmodified chitosan,33 suggesting that TLC would be more positively charged than the unmodified chitosan.

Bottom Line: To develop a chitosan-based nonviral gene carrier capable of delivering genes specifically into hepatoma cells, a bifunctional peptide composed of the TAT (transactivator of transcription) peptide and luteinizing hormone-releasing hormone (LHRH) was conjugated with low molecular weight chitosan, resulting in a TAT-LHRH-chitosan conjugate (TLC).In vitro targeting specificity and transfection efficiency were analyzed with a GE IN Cell Analyzer 2000 High-Content Cellular Analysis System.The results demonstrated that TLC had stronger DNA condensing power than unmodified chitosan, and that TLCDNPs were of roughly round shape with average diameter of 70-85 nm and zeta potential of +30 mV and were relatively stable in solution.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bioengineering, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin Key Laboratory of Biomedical Materials, Tianjin, People's Republic of China.

ABSTRACT
To develop a chitosan-based nonviral gene carrier capable of delivering genes specifically into hepatoma cells, a bifunctional peptide composed of the TAT (transactivator of transcription) peptide and luteinizing hormone-releasing hormone (LHRH) was conjugated with low molecular weight chitosan, resulting in a TAT-LHRH-chitosan conjugate (TLC). TLC/DNA nanoparticles (TLCDNPs) were characterized by agarose gel retardation, atomic force microscopy, and dynamic light scattering analysis. In vitro targeting specificity and transfection efficiency were analyzed with a GE IN Cell Analyzer 2000 High-Content Cellular Analysis System. The results demonstrated that TLC had stronger DNA condensing power than unmodified chitosan, and that TLCDNPs were of roughly round shape with average diameter of 70-85 nm and zeta potential of +30 mV and were relatively stable in solution. The in vitro study demonstrated TLC was highly selective for hepatoma cells and essentially nontoxic.

Show MeSH
Related in: MedlinePlus