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Stability, Intracellular Delivery, and Release of siRNA from Chitosan Nanoparticles Using Different Cross-Linkers.

Raja MA, Katas H, Jing Wen T - PLoS ONE (2015)

Bottom Line: The resulting nanoparticles were compared with regard to their physicochemical properties including particle size, zeta potential, morphology, binding and encapsulation efficiencies.The cellular uptake studies with CS-TPP-siRNA nanoparticles showed successful delivery of siRNA within cytoplasm of DLD-1 cells.The results demonstrate that ionically cross-linked CS-TPP nanoparticles are biocompatible non-viral gene delivery system and generate a solid ground for further optimization studies, for example with regard to steric stabilization and targeting.

View Article: PubMed Central - PubMed

Affiliation: Centre for Drug Delivery Research, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, Kuala Lumpur, Malaysia.

ABSTRACT
Chitosan (CS) nanoparticles have been extensively studied for siRNA delivery; however, their stability and efficacy are highly dependent on the types of cross-linker used. To address this issue, three common cross-linkers; tripolyphosphate (TPP), dextran sulphate (DS) and poly-D-glutamic acid (PGA) were used to prepare siRNA loaded CS-TPP/DS/PGA nanoparticles by ionic gelation method. The resulting nanoparticles were compared with regard to their physicochemical properties including particle size, zeta potential, morphology, binding and encapsulation efficiencies. Among all the formulations prepared with different cross linkers, CS-TPP-siRNA had the smallest particle size (ranged from 127 ± 9.7 to 455 ± 12.9 nm) with zeta potential ranged from +25.1 ± 1.5 to +39.4 ± 0.5 mV, and high entrapment (>95%) and binding efficiencies. Similarly, CS-TPP nanoparticles showed better siRNA protection during storage at 4˚C and as determined by serum protection assay. TEM micrographs revealed the assorted morphology of CS-TPP-siRNA nanoparticles in contrast to irregular morphology displayed by CS-DS-siRNA and CS-PGA-siRNA nanoparticles. All siRNA loaded CS-TPP/DS/PGA nanoparticles showed initial burst release followed by sustained release of siRNA. Moreover, all the formulations showed low and concentration-dependent cytotoxicity with human colorectal cancer cells (DLD-1), in vitro. The cellular uptake studies with CS-TPP-siRNA nanoparticles showed successful delivery of siRNA within cytoplasm of DLD-1 cells. The results demonstrate that ionically cross-linked CS-TPP nanoparticles are biocompatible non-viral gene delivery system and generate a solid ground for further optimization studies, for example with regard to steric stabilization and targeting.

No MeSH data available.


Related in: MedlinePlus

Internalization and localization of 6-FAM labelled siRNA-loaded CS-TPP nanoparticles in DLD-1 cells after 4 h incubation.Control: (a) naked 6-FAM labelled siRNA, (b) nuclear staining with Hoechst 33342 (blue), (c) an overlay of (a)-(b). Nanoparticles: (d) CS-TPP nanoparticles loaded with 6-FAM labelled siRNA (green), (e) nuclear staining with Hoechst 33342 (blue), (f) an overlay of (d)-(e). Scale bar represents 10μm.
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pone.0128963.g010: Internalization and localization of 6-FAM labelled siRNA-loaded CS-TPP nanoparticles in DLD-1 cells after 4 h incubation.Control: (a) naked 6-FAM labelled siRNA, (b) nuclear staining with Hoechst 33342 (blue), (c) an overlay of (a)-(b). Nanoparticles: (d) CS-TPP nanoparticles loaded with 6-FAM labelled siRNA (green), (e) nuclear staining with Hoechst 33342 (blue), (f) an overlay of (d)-(e). Scale bar represents 10μm.

Mentions: This study was performed to determine whether CS-TPP nanoparticles facilitated the delivery of siRNA into DLD-1 cells. Fluorescein-labelled siRNA (6-FAM-siRNA) was used in this study. 6-FAM-siRNA-loaded CS-TPP nanoparticles were incubated with DLD-1 cells for 4h in the presence of 10% FBS. CS-TPP-siRNA nanoparticles infiltrated cells and were primarily distributed in the cytoplasm, which is in agreement with previous observations[40] (Fig 10D–10F). However, naked 6-FAM-siRNA was not detected (Fig 10A–10C). Fluorescence from 6-FAM-siRNA-loaded CS-TPP nanoparticles was detected in the cytoplasm suggesting that siRNA was dissociated from CS-TPP nanoparticles.


Stability, Intracellular Delivery, and Release of siRNA from Chitosan Nanoparticles Using Different Cross-Linkers.

Raja MA, Katas H, Jing Wen T - PLoS ONE (2015)

Internalization and localization of 6-FAM labelled siRNA-loaded CS-TPP nanoparticles in DLD-1 cells after 4 h incubation.Control: (a) naked 6-FAM labelled siRNA, (b) nuclear staining with Hoechst 33342 (blue), (c) an overlay of (a)-(b). Nanoparticles: (d) CS-TPP nanoparticles loaded with 6-FAM labelled siRNA (green), (e) nuclear staining with Hoechst 33342 (blue), (f) an overlay of (d)-(e). Scale bar represents 10μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4465927&req=5

pone.0128963.g010: Internalization and localization of 6-FAM labelled siRNA-loaded CS-TPP nanoparticles in DLD-1 cells after 4 h incubation.Control: (a) naked 6-FAM labelled siRNA, (b) nuclear staining with Hoechst 33342 (blue), (c) an overlay of (a)-(b). Nanoparticles: (d) CS-TPP nanoparticles loaded with 6-FAM labelled siRNA (green), (e) nuclear staining with Hoechst 33342 (blue), (f) an overlay of (d)-(e). Scale bar represents 10μm.
Mentions: This study was performed to determine whether CS-TPP nanoparticles facilitated the delivery of siRNA into DLD-1 cells. Fluorescein-labelled siRNA (6-FAM-siRNA) was used in this study. 6-FAM-siRNA-loaded CS-TPP nanoparticles were incubated with DLD-1 cells for 4h in the presence of 10% FBS. CS-TPP-siRNA nanoparticles infiltrated cells and were primarily distributed in the cytoplasm, which is in agreement with previous observations[40] (Fig 10D–10F). However, naked 6-FAM-siRNA was not detected (Fig 10A–10C). Fluorescence from 6-FAM-siRNA-loaded CS-TPP nanoparticles was detected in the cytoplasm suggesting that siRNA was dissociated from CS-TPP nanoparticles.

Bottom Line: The resulting nanoparticles were compared with regard to their physicochemical properties including particle size, zeta potential, morphology, binding and encapsulation efficiencies.The cellular uptake studies with CS-TPP-siRNA nanoparticles showed successful delivery of siRNA within cytoplasm of DLD-1 cells.The results demonstrate that ionically cross-linked CS-TPP nanoparticles are biocompatible non-viral gene delivery system and generate a solid ground for further optimization studies, for example with regard to steric stabilization and targeting.

View Article: PubMed Central - PubMed

Affiliation: Centre for Drug Delivery Research, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, Kuala Lumpur, Malaysia.

ABSTRACT
Chitosan (CS) nanoparticles have been extensively studied for siRNA delivery; however, their stability and efficacy are highly dependent on the types of cross-linker used. To address this issue, three common cross-linkers; tripolyphosphate (TPP), dextran sulphate (DS) and poly-D-glutamic acid (PGA) were used to prepare siRNA loaded CS-TPP/DS/PGA nanoparticles by ionic gelation method. The resulting nanoparticles were compared with regard to their physicochemical properties including particle size, zeta potential, morphology, binding and encapsulation efficiencies. Among all the formulations prepared with different cross linkers, CS-TPP-siRNA had the smallest particle size (ranged from 127 ± 9.7 to 455 ± 12.9 nm) with zeta potential ranged from +25.1 ± 1.5 to +39.4 ± 0.5 mV, and high entrapment (>95%) and binding efficiencies. Similarly, CS-TPP nanoparticles showed better siRNA protection during storage at 4˚C and as determined by serum protection assay. TEM micrographs revealed the assorted morphology of CS-TPP-siRNA nanoparticles in contrast to irregular morphology displayed by CS-DS-siRNA and CS-PGA-siRNA nanoparticles. All siRNA loaded CS-TPP/DS/PGA nanoparticles showed initial burst release followed by sustained release of siRNA. Moreover, all the formulations showed low and concentration-dependent cytotoxicity with human colorectal cancer cells (DLD-1), in vitro. The cellular uptake studies with CS-TPP-siRNA nanoparticles showed successful delivery of siRNA within cytoplasm of DLD-1 cells. The results demonstrate that ionically cross-linked CS-TPP nanoparticles are biocompatible non-viral gene delivery system and generate a solid ground for further optimization studies, for example with regard to steric stabilization and targeting.

No MeSH data available.


Related in: MedlinePlus