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New methodologies to characterize the effectiveness of the gene transfer mediated by DNA-chitosan nanoparticles.

Centelles MN, Qian C, Campanero MA, Irache JM - Int J Nanomedicine (2008)

Bottom Line: No significant influence of MW was observed on the levels of luciferase expression.Nevertheless, the same administration procedure of the three formulations did not improve the levels of transgene expression obtained with naked DNA.This fact could be explained by the rapid physiological turn-over of enterocytes and by the ability of chitosan nanoparticles to control the DNA release.

View Article: PubMed Central - PubMed

Affiliation: Centro Galénico, Departamento Farmacia y Tecnología Farmacéutica, University of Navarra, Pamplona, Spain.

ABSTRACT
In this work three DNA-chitosan nanoparticle formulations (Np), differing in the molecular weight (MW; 150 kDa, 400 kDa, and 600 kDa) of the polysaccharide, were prepared and administered by two different administration routes: the hydrodynamics-based procedure and the intraduodenal injection. After the hydrodynamic injection, DNA-chitosan nanoparticles were predominantly accumulated in the liver, where the transgene was expressed during at least 105 days. No significant influence of MW was observed on the levels of luciferase expression. The curves of bioluminescence versus time obtained using the charge-coupled device (CCD) camera were described and divided in three phases: (i) the initial phase, (ii) the sustained release step and (iii) the decline phase (promotor inactivation, immunological and physiological processes). From these curves, which describe the transgene expression profile, the behavior of the different formulations as gene delivery systems was characterized. Therefore, the following parameters such as C(max) (maximum level of detected bioluminescence), AUC (area under the bioluminescence-time curve) and MET (mean time of the transgene expression) were calculated. This approach offers the possibility of studying and comparing transgene expression kinetics among a wide variety of gene delivery systems. Finally, the intraduodenal administration of naked DNA permitted the gene transfer in a dose dependent manner quantifiable with the CCD camera within 3 days. Nevertheless, the same administration procedure of the three formulations did not improve the levels of transgene expression obtained with naked DNA. This fact could be explained by the rapid physiological turn-over of enterocytes and by the ability of chitosan nanoparticles to control the DNA release.

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(A) Intestinal sections expressing GFP, 24 h post intraduodenal administration. (A1) 1 ml of saline as control, (A2) Np400 corresponding to a dose of 50 μg plasmid (pEGFP) in 1 ml of formulation. (B) Bioluminescent imaging of intestinal luciferase expression in living mice using a cooled CCD camera. (B1) 25 μg naked DNA in a volume of 500 μl saline; (B2) 50 μg naked DNA in a volume of 500 μl saline; (B3) 50 μg naked DNA in a volume of 1 ml saline; (B4) 100 μg naked DNA in a volume of 1 ml saline. (B5) Untreated animal. (B6) Amount of Np150 corresponding to 50 μg of DNA in 1 ml saline (B7) Np400 corresponding to 50 μg of DNA in 1 ml saline, and (B8) Np600 corresponding to 50 μg of DNA in 1 ml saline.
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f4-ijn-3-451: (A) Intestinal sections expressing GFP, 24 h post intraduodenal administration. (A1) 1 ml of saline as control, (A2) Np400 corresponding to a dose of 50 μg plasmid (pEGFP) in 1 ml of formulation. (B) Bioluminescent imaging of intestinal luciferase expression in living mice using a cooled CCD camera. (B1) 25 μg naked DNA in a volume of 500 μl saline; (B2) 50 μg naked DNA in a volume of 500 μl saline; (B3) 50 μg naked DNA in a volume of 1 ml saline; (B4) 100 μg naked DNA in a volume of 1 ml saline. (B5) Untreated animal. (B6) Amount of Np150 corresponding to 50 μg of DNA in 1 ml saline (B7) Np400 corresponding to 50 μg of DNA in 1 ml saline, and (B8) Np600 corresponding to 50 μg of DNA in 1 ml saline.

Mentions: Figure 4A shows the GFP expression in two distant sections of the intestine (duodenum and ileum) 24 hours post administration of DNA loaded in Np400. These photographs show that our nanoparticles were able to transduce the enterocyte monolayer along the intestinal tract, and with high intensity in some sections. These data confirmed the great expectative projected by several studies previously published (Roy et al 1999; Cheng et al 2004; Guliyeva et al 2006; MacLaughlin et al 1998).


New methodologies to characterize the effectiveness of the gene transfer mediated by DNA-chitosan nanoparticles.

Centelles MN, Qian C, Campanero MA, Irache JM - Int J Nanomedicine (2008)

(A) Intestinal sections expressing GFP, 24 h post intraduodenal administration. (A1) 1 ml of saline as control, (A2) Np400 corresponding to a dose of 50 μg plasmid (pEGFP) in 1 ml of formulation. (B) Bioluminescent imaging of intestinal luciferase expression in living mice using a cooled CCD camera. (B1) 25 μg naked DNA in a volume of 500 μl saline; (B2) 50 μg naked DNA in a volume of 500 μl saline; (B3) 50 μg naked DNA in a volume of 1 ml saline; (B4) 100 μg naked DNA in a volume of 1 ml saline. (B5) Untreated animal. (B6) Amount of Np150 corresponding to 50 μg of DNA in 1 ml saline (B7) Np400 corresponding to 50 μg of DNA in 1 ml saline, and (B8) Np600 corresponding to 50 μg of DNA in 1 ml saline.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2636586&req=5

f4-ijn-3-451: (A) Intestinal sections expressing GFP, 24 h post intraduodenal administration. (A1) 1 ml of saline as control, (A2) Np400 corresponding to a dose of 50 μg plasmid (pEGFP) in 1 ml of formulation. (B) Bioluminescent imaging of intestinal luciferase expression in living mice using a cooled CCD camera. (B1) 25 μg naked DNA in a volume of 500 μl saline; (B2) 50 μg naked DNA in a volume of 500 μl saline; (B3) 50 μg naked DNA in a volume of 1 ml saline; (B4) 100 μg naked DNA in a volume of 1 ml saline. (B5) Untreated animal. (B6) Amount of Np150 corresponding to 50 μg of DNA in 1 ml saline (B7) Np400 corresponding to 50 μg of DNA in 1 ml saline, and (B8) Np600 corresponding to 50 μg of DNA in 1 ml saline.
Mentions: Figure 4A shows the GFP expression in two distant sections of the intestine (duodenum and ileum) 24 hours post administration of DNA loaded in Np400. These photographs show that our nanoparticles were able to transduce the enterocyte monolayer along the intestinal tract, and with high intensity in some sections. These data confirmed the great expectative projected by several studies previously published (Roy et al 1999; Cheng et al 2004; Guliyeva et al 2006; MacLaughlin et al 1998).

Bottom Line: No significant influence of MW was observed on the levels of luciferase expression.Nevertheless, the same administration procedure of the three formulations did not improve the levels of transgene expression obtained with naked DNA.This fact could be explained by the rapid physiological turn-over of enterocytes and by the ability of chitosan nanoparticles to control the DNA release.

View Article: PubMed Central - PubMed

Affiliation: Centro Galénico, Departamento Farmacia y Tecnología Farmacéutica, University of Navarra, Pamplona, Spain.

ABSTRACT
In this work three DNA-chitosan nanoparticle formulations (Np), differing in the molecular weight (MW; 150 kDa, 400 kDa, and 600 kDa) of the polysaccharide, were prepared and administered by two different administration routes: the hydrodynamics-based procedure and the intraduodenal injection. After the hydrodynamic injection, DNA-chitosan nanoparticles were predominantly accumulated in the liver, where the transgene was expressed during at least 105 days. No significant influence of MW was observed on the levels of luciferase expression. The curves of bioluminescence versus time obtained using the charge-coupled device (CCD) camera were described and divided in three phases: (i) the initial phase, (ii) the sustained release step and (iii) the decline phase (promotor inactivation, immunological and physiological processes). From these curves, which describe the transgene expression profile, the behavior of the different formulations as gene delivery systems was characterized. Therefore, the following parameters such as C(max) (maximum level of detected bioluminescence), AUC (area under the bioluminescence-time curve) and MET (mean time of the transgene expression) were calculated. This approach offers the possibility of studying and comparing transgene expression kinetics among a wide variety of gene delivery systems. Finally, the intraduodenal administration of naked DNA permitted the gene transfer in a dose dependent manner quantifiable with the CCD camera within 3 days. Nevertheless, the same administration procedure of the three formulations did not improve the levels of transgene expression obtained with naked DNA. This fact could be explained by the rapid physiological turn-over of enterocytes and by the ability of chitosan nanoparticles to control the DNA release.

Show MeSH