Introduction: Alzheimer’s disease (AD) is the most common type of dementia, representing over 40 million people diagnosed worldwide[1]. Recent studies indicate that growth factors (GFs) found in the mature brain, such as bone morphogenetic proteins 9 (BMP-9), have a great therapeutic potential[2]-[4]. However, BMPs are big, expensive to produce and they face many hurdles in their addressing to the brain such as the blood brain barrier. We have developed small and low cost peptides derived from BMP-9 (pBMP-9, SpBMP-9)[5] that can be encapsulated into chitosan nanoparticle (CNPs) for intranasal injection. CNPs are getting uprising attention because of their biocompatibility and mucoadhesive properties[6]-[8]. This study reports the effect of pBMP-9 and SpBMP-9 compared to BMP-9 on the differentiation of human neuroblastoma cells SH-SY5Y and their encapsulation in CNPs.
Materials and Methods: Cells were stimulated with equimolar concentrations of BMP-9, pBMP-9 and SpBMP-9 with or without retinoic acid (RA). Kinetics of signaling pathways activation (Smad, MAPK, Akt) were assessed by western blotting. Morphology analyses and average neurite length was determined from phase contrast images with an image analysis program (MatLab). The expression of neuronal differentiation markers (βIII-tubulin, MAP-2, NeuN, NSE and VAchT) was assessed by Western blotting and immunolabeling. The synthesis of acetylcholine (Ach) was measured by fluorescence detection using Amplex® Red acetylcholine/acetylcholinesterase assay Kit. CNPs synthesis and loading was achieved by ionotropic gelation by mixing solutions of chitosan plus peptide derived from BMP-9 and tripolyphosphate. CNPs were recovered by centrifugation and freeze dried. CNPs size and peptide encapsulation efficiency were evaluated with scanning electron microscopy and high-performance liquid chromatography respectively.
Results and Discussion: BMP-9, pBMP-9 and SpBMP-9 activated similarly canonical Smad, MAPK and Akt pathways, with a faster response with RA. Interestingly, at 5 days both pBMP-9 and SpBMP-9 increased the number of neurites and their length compared with BMP-9 and control with or without RA. As observed by the levels of neuronal markers, the presence of RA alone could increase the SH-SY5Y differentiation, but adding pBMP-9 and SpBMP-9, unlike BMP-9 enhanced this differentiation. In addition, pBMP-9 and SpBMP-9 increased both the amount of VAchT and the production of Ach. CNPs synthesis gave particles with sizes around few hundred nanometers. Both peptides were successfully encapsulated into CNPs.
Conclusion: Those results show that pBMP-9 and SpBMP-9 seems to have a more potent action in terms of neuronal differentiation compared to BMP-9. Their capacity to be encapsulated in CNPs and their small size make them prone to be used as a therapeutic strategy for addressing GFs to the brain. However, further investigations are required to determine the optimal release system.
References:
[1] World Health Organization, Dementia, A public Health Priority, Report 2012, Internet: www.who.int (consulted in november 2014)
[2] Lauzon M.A., Daviau A., Marcos B., Faucheux N., Growth factor treatment to overcome Alzheimer’s dysfunctional signaling, Cell Signaling, 27(6), 2015, p. 1025-1038
[3] Burke R. M., Norman T. A., Haydar T. F., Slack B. E., Leeman S. E., Krzysztof B., Mellott T., BMP9 ameliorates amyloidosis and the cholinergic defect in a mouse model of Alzheimer’s disease, PNAS, 110(48), 2013, p.19567-19572
[4] Lopez-Coviella I., Berse B., Krauss R., Thies R. S., Blusztajn J.K., Induction and maintenance of the neuronal cholinergic phenotype in the central nervous system by BMP-9, Science, 289, 2000, p.313-316
[5] Bergeron E., Leblanc E., Drevelle O., Giguère R., Beauvais S., Grenier G., Faucheux N., The evaluation of ectopic bone formation induced by delivery systems ant their mathematical modeling, Tissue Engineering Part A, 2012, 18(3-4), p. 342-352
[6] Nagpal K., Singh S.K., Mishra D. N., Chitosan nanoparticles: a promising system in novel drug delivery, Chemical and Pharmaceutical Bulletin, 2010, 58(11), p. 1423-1430
[7] Sarvaiya J. and Agrawal Y. K., Chitosan as a suitable nanocarrier material for anti-Alzheimer drug delivery, International Journal of Biological Macromolecules, 2015, 72, p. 454-465
[8] Aspden T.J., Mason J. D., Jones N. S., Lowe J., Skaugrud Ø., Illum L., Chitosan as a nasal delivery system: the effect of chitosan solutions on in vitro and in vivo mucociliary transport rates in human turbinates and volunteers, Journal of Pharmaceutical Sciences, 1997, 86(4), p. 509-513