Publications in January 2015
At the beginning of the year 2015, an incredible large number of chitosan-related articles have been released, indicating another record year. All in all, 196 publications about chitosan and its derivatives were contributed by scientist from all over the world, especially from China (46 articles), India (29) and USA (15). The main terms address chitosan in evaluation studies, nanoparticles and pharmaceutical preparations.
|International journal of biological macromolecules||34|
|Colloids and surfaces. B, Biointerfaces||6|
|Materials science & engineering. C, Materials for biological applications||6|
Table: Leading scientific journals in the field of chitosan research and the number of publications in January 2015. Source: GoPubMed
Over ten years ago, chitosan initially emerged as an alternative for non-viral gene delivery systems. Meanwhile, numerous studies analysed transfection efficiency, vehicle stability and cytotoxicity of distinct chitosan formulations. Two novel scientific approaches are described below. They offer new solutions for major issues of gene transfection, improving transfection efficiency for primary cells and transfer rate of large gene sequences.
Spontaneous gene transfection of human bone cells using 3D mineralized alginate-chitosan macrocapsules.
Green D.W., Kim E.J., Jung H.S.; Journal of biomedical materials research. Part A. doi:10.1002/jbm.a. 35414.; January 2015
The transfection of human cell lines by non-viral gene transfections strategies is often restricted by their low transfection efficiency and high toxicity compared to viral-based methods. The authors of this study analysed whether a 3D chitosan scaffold can improve transfection efficiency for cell lines and primary cells. The 3D capsule-based transfection system was composed of alginate beads, surrounded by an outer shell of chitosan and calcium phosphate. Calcium phosphate served as transfection reagent. Alginate and chitosan embedded plasmid DNA and cells for transfection. The capsules were loaded with different plasmids carrying lacZ, gfp or Sox-9 genes. Human osteosarcoma cell lines Saos-2, MG63 and primary human bone marrow stromal cells (hBMSCs) were chosen as transfection cell model.
Results for mineralized alginate-chitosan macrocapsules:
- Low cytotoxicity compared to lipofection or nucleofection
- High transfection efficiency: ≤ 65%
- in human cell lines: Saos-2, MG63
- in primary human bone marrow stromal cells (hBMSCs)
- High self-transfection efficiency of hBMSCs: ≤ 90%
- Sox-9 expression over 7 days
- Highest transfection efficiency for two-layer capsules
- plasmid-containing guest structure is incorporated into cell-loaded host bead
Conclusion: Macrocapsules generated from mineralized polysaccharides display excellent vehicle properties for gene delivery, while chitosan-free or unmineralized capsules were ineffective for transfection. The 3D scaffold clearly improved the reliability and reproducibility of gene transfection, especially for primary cells, as their transfection is often challenging.
Enhancement of chitosan-mediated gene delivery through combination with phiC31 integrase.
Oliveira A.V., Silva G.A., Chung D.C.; Acta Biomaterialia. pii: S1742-7061(15)00014-8. doi: 10.1016/j.actbio.2015.01.013. [Epub ahead of print], January 2015
The authors of the present study invented a non-viral gene delivery system based on chitosan polyplexes and phiC31-integrase.
Chitosan-based polyplexes were prepared from equal amounts of chitosan solutions (1 mg/ml, pH 5.5) and sodium sulfate solution (25 mM). The chitosan polyplexes (80 or 260 kDa) were combined with DNA-plasmids (pDNA) carrying the reference genes like gfp or cep290. Both plasmids were constructed with attachment sites for the phi31 integrase, a protein, which was encoded by a third plasmid. To load polyplexes with pDNA, 250 µg chitosan was mixed with 26.5 µg DNA. The transfection was performed with HEK293 cells.
- Stable and side-specific gene integration
- Delivery of large genes sequences (> 8 Kb) Exceeding capacity of adeno-associated virus vectors
- Sustained gene expression
- GFP (1 Kb) expression over 16 weeks
- CEP290 (8 Kb) expression over 6 weeks
- Greater transfection efficiency for polyplexes with low MW (80 kDa)
- All formulation of chitosan polyplexes possess similar: DNA complexation efficiency, spherical morphology and polydispersity
- Transfection efficiency and transgene expression varied with polymer type and polyplexe size
Conclusion: A gene delivery system composed of chitosan polyplexes and phiC31 integrase significantly improves transfection efficiency for large genes (> 8 Kb), allows a side-specific integration and is stable over at least 6 weeks. Furthermore, it displays no cytotoxicity, which makes it superior to virus-based transfection strategies.