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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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Electrophoresis of chitosan-DNA nanoparticles to determine effective encapsulation, DNAse I protection (digestion with DNAse I), plasmid integrity following release (digestion with chitosanase), and release after charge interaction disruption with NaOH or FBS. (A) Lanes 1 and 16: Molecular weight marker VII; Lane 2: pX2-Luc plasmid DNA; Lane 3: + DNAse I; Lane 4-8-12: nanoparticles composed of 150, 400 or 600 kDa chitosan respectively; Lane 5-9-13: + DNAse I; Lane 6-10-14: + chitosanase; Lane 7-11-15: + NaOH. (B) Nanoparticles composed of 150, 400, or 600 kDa chitosan incubated 2 h (37 °C) with FBS at untreated 20%, 10%, 5%, and 2.5%, respectively.
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f2-ijn-3-451: Electrophoresis of chitosan-DNA nanoparticles to determine effective encapsulation, DNAse I protection (digestion with DNAse I), plasmid integrity following release (digestion with chitosanase), and release after charge interaction disruption with NaOH or FBS. (A) Lanes 1 and 16: Molecular weight marker VII; Lane 2: pX2-Luc plasmid DNA; Lane 3: + DNAse I; Lane 4-8-12: nanoparticles composed of 150, 400 or 600 kDa chitosan respectively; Lane 5-9-13: + DNAse I; Lane 6-10-14: + chitosanase; Lane 7-11-15: + NaOH. (B) Nanoparticles composed of 150, 400, or 600 kDa chitosan incubated 2 h (37 °C) with FBS at untreated 20%, 10%, 5%, and 2.5%, respectively.

Mentions: The next objective of our study was to evaluate the integrity of DNA after the preparative process, its release from the chitosan nanoparticles as well as the protection offered by these carriers to the loaded plasmid. Therefore, gel electrophoresis experiments were performed (Figure 2). Loaded DNA-chitosan nanoparticles displayed a pattern of bands co-localized with the loading well, meaning that encapsulated DNA was unable to migrate into the gel due to the strong interactions with the different chitosans (Figure 2A; Lanes 4, 8, and 12). This ionic interaction was independent from the molecular weight of the polysaccharide. The protection of DNA was verified with DNAse I as model enzyme. Naked DNA showed a complete degradation (Figure 2A; lane 3), whereas encapsulated DNA was preserved (at the concentration used) into the loading wells without any longer migration (Figure 2A; lanes 5, 9, and 13). Furthermore, when nanoparticles were digested with chitosanase, the released DNA displayed the same electrophoretic migration pattern as the naked DNA (Figure 2A; lanes 6, 10, 14, and 2). This result confirmed that the preparative process of nanoparticles preserved the integrity of DNA. This result agrees well with previous observations reported by Mao and colleagues (2001). To reinforce the fact that the release of DNA can be achieved by charge interaction disruption, nanoparticles were incubated with either NaOH or FBS. In the first case, the released DNA showed a retarded electrophoretic mobility into the gel, indicating that interaction disruption in our experimental conditions were only partial and chitosan molecules remained bound to DNA (Figure 2A; lanes 7, 11, and 15). In the second case, the incubation between nanoparticles and FBS for 2 h led to a partial release of DNA with an enhanced electrophoretic mobility (Figure 2B). These results point out the strong interaction between DNA and chitosan by means of ionic interactions and confirmed the data described by Liu and colleagues (2005).


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)

Electrophoresis of chitosan-DNA nanoparticles to determine effective encapsulation, DNAse I protection (digestion with DNAse I), plasmid integrity following release (digestion with chitosanase), and release after charge interaction disruption with NaOH or FBS. (A) Lanes 1 and 16: Molecular weight marker VII; Lane 2: pX2-Luc plasmid DNA; Lane 3: + DNAse I; Lane 4-8-12: nanoparticles composed of 150, 400 or 600 kDa chitosan respectively; Lane 5-9-13: + DNAse I; Lane 6-10-14: + chitosanase; Lane 7-11-15: + NaOH. (B) Nanoparticles composed of 150, 400, or 600 kDa chitosan incubated 2 h (37 °C) with FBS at untreated 20%, 10%, 5%, and 2.5%, respectively.
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Related In: Results  -  Collection

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

f2-ijn-3-451: Electrophoresis of chitosan-DNA nanoparticles to determine effective encapsulation, DNAse I protection (digestion with DNAse I), plasmid integrity following release (digestion with chitosanase), and release after charge interaction disruption with NaOH or FBS. (A) Lanes 1 and 16: Molecular weight marker VII; Lane 2: pX2-Luc plasmid DNA; Lane 3: + DNAse I; Lane 4-8-12: nanoparticles composed of 150, 400 or 600 kDa chitosan respectively; Lane 5-9-13: + DNAse I; Lane 6-10-14: + chitosanase; Lane 7-11-15: + NaOH. (B) Nanoparticles composed of 150, 400, or 600 kDa chitosan incubated 2 h (37 °C) with FBS at untreated 20%, 10%, 5%, and 2.5%, respectively.
Mentions: The next objective of our study was to evaluate the integrity of DNA after the preparative process, its release from the chitosan nanoparticles as well as the protection offered by these carriers to the loaded plasmid. Therefore, gel electrophoresis experiments were performed (Figure 2). Loaded DNA-chitosan nanoparticles displayed a pattern of bands co-localized with the loading well, meaning that encapsulated DNA was unable to migrate into the gel due to the strong interactions with the different chitosans (Figure 2A; Lanes 4, 8, and 12). This ionic interaction was independent from the molecular weight of the polysaccharide. The protection of DNA was verified with DNAse I as model enzyme. Naked DNA showed a complete degradation (Figure 2A; lane 3), whereas encapsulated DNA was preserved (at the concentration used) into the loading wells without any longer migration (Figure 2A; lanes 5, 9, and 13). Furthermore, when nanoparticles were digested with chitosanase, the released DNA displayed the same electrophoretic migration pattern as the naked DNA (Figure 2A; lanes 6, 10, 14, and 2). This result confirmed that the preparative process of nanoparticles preserved the integrity of DNA. This result agrees well with previous observations reported by Mao and colleagues (2001). To reinforce the fact that the release of DNA can be achieved by charge interaction disruption, nanoparticles were incubated with either NaOH or FBS. In the first case, the released DNA showed a retarded electrophoretic mobility into the gel, indicating that interaction disruption in our experimental conditions were only partial and chitosan molecules remained bound to DNA (Figure 2A; lanes 7, 11, and 15). In the second case, the incubation between nanoparticles and FBS for 2 h led to a partial release of DNA with an enhanced electrophoretic mobility (Figure 2B). These results point out the strong interaction between DNA and chitosan by means of ionic interactions and confirmed the data described by Liu and colleagues (2005).

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