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Nanostructured microspheres for the controlled release of BMP-7 and the suppression of glioblastoma initiating cells

P. Gonzalez-Gomez, Instituto de Salud Carlos III, Spain; E. Reguera-Nuñez, Universidad de Santiago de Compostela, Spain; J. Crecente-Campo, Universidad de Santiago de Compostela, Spain; C. Zahonero, Instituto de Salud Carlos III, Spain; A. Hernandez-Lain, Hospital 12 de Octubre, Spain; H. Mira, Instituto de Salud Carlos III, Spain; P. Sanchez-Gomez, Instituto de Salud Carlos III, Spain; M. Garcia-Fuentes, Universidad de Santiago de Compostela, Spain

Glioblastoma initiating cells (GICs) are responsible for high-rates of tumor recurrence since they present high resistance to conventional chemotherapy and capacity to initiate and sustain tumor growth [1]. Bone morphogenetic proteins (BMPs) have been identified as signaling proteins capable of inhibiting the tumor initiation properties of GICs, thereby suppressing tumor recurrence [2]. Unfortunately, BMP-based therapies are limited in practice by the short half-life of this protein (~5 min) and its lack of permeability through the blood-brain-barrier. To overcome these limitations our team aimed at designing a microsphere system loaded with BMP-7 for implantation in the central nervous system. The medical strategy would be to implant these microspheres at the time of primary tumor resection to provide sustained supply of BMP-7 at the tumor site for several weeks.
Proteins are incompatible with most biodegradable controlled release polymers, and frequently degrade during encapsulation and release. To prevent these undesirable processes we took advantage of the specific affinity of BMP-7 for glycosaminoglycans, and complexed this protein with heparin, and then coated this nanostructure with poloxamine, a cationic polyethylene glycol derivative [3]. The nanocomplexes of BMP-7/heparin/poloxamine could be freeze-dried and resuspended in an organic solvent, to be efficiently encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres through a O/O solvent emulsification method. The microspheres prepared by this method had 80 μm in particle size, regular morphology, and often a hollow core. BMP-7 encapsulation in the microspheres was high (>85%) as quantified by ELISA. Release studies confirmed over 60 days of BMP-7 controlled release from the microspheres with sustained kinetics. Importantly, the released protein was in an antigenically active conformation. Scanning microscopy studies confirmed that BMP-7 release was related to microsphere erosion as it hydrolyzed in buffer at 37ºC. In vitro studies performed in a U87MG neurosphere model showed that released BMP-7, even that from the 90 days time-point, is bioactive and capable of suppressing the capacity of these cells for sphere formation [4].
To validate the therapeutic concept we decided to test the microspheres in vivo, using human primary GIC cell lines. We tested eight different primary cell lines and we found that BMP-7 was able to inhibit tumor formation in those expressing its functional receptor (i.e. BMPR1B). We tested the capacity of the microspheres for inhibiting tumor formation in a xenograft model, using a BMP-responsive primary GIC cell line. This in vivo experiment confirmed the therapeutic potential of the microspheres loaded with BMP-7: the tumors formed in the back of the mice treated with the microspheres were significantly smaller than those of the controls. Inhibition of tumor formation in the study was linked to activation of the BMP signaling cascade. Moreover, the tumors treated with the microspheres showed reduced expression of stem cell markers typical of tumor malignancy, and up-regulation of selected tumor suppressors [5]. We envisage that this kind of selective therapy for tumor initiating cells could have a synergistic effect in combination with conventional cytoreductive therapy or with immunotherapy.

1. Chen, J.; Li Y.; Yu T-S.; McKay R.; Burns, D.; Kernie, S.; Parada, L.F., A Restricted Cell Population Propagates Glioblastoma Growth after Chemotherapy, Nature 488, 522–526 (2012).
2. Piccirillo, SGM; Reynolds, BA; Zanetti, N.; Lamorte, G.; Binda, E.; Broggi, G.; Brem, H.; Olivi, A.; Dimeco, F.; Vescovi, AL., Bone Morphogenetic Proteins Inhibit the Tumorigenic Potential of Human Brain Tumour-Initiating Cells, Nature 444, 761–765 (2006).
3. García Fuentes, M.; Reguera Núñez, E.; Csaba, N., Controlled release formulation, PCT/ES2013/070655.
4. Reguera-Nuñez, E.; Roca, C.; Hardy, E.; Csaba, N.; de la Fuente, M.; Garcia-Fuentes, M., Implantable controlled release devices for BMP-7 delivery and suppression of glioblastoma initiating cells, Biomaterials 35, 2859-2867 (2014).
5. González-Gómez, P.; Crecente-Campo, J.; Zahonero, C.;de la Fuente, M.; Hernández-Laín, A.; Mira, H.; Sánchez-Gómez, P.; Garcia-Fuentes, M. Controlled release microspheres loaded with BMP7 suppress primary tumors from human glioblastoma, Oncotarget (2015, in press).

This study was supported by grants from: Ministerio de Economía y Competitividad, Fondo de Investigación Sanitaria (PI12/101 to HM; PI12/00775 to PSG; PS09/1786 to MGF and PI13/01258 to AHL), Comunidad de Madrid (S2010/BMD-2336 to HM), Xunta de Galicia (EM2013/042 to MGF), Fundación BBVA (2014-PO010 to MGF) and Ministerio de Economía y Competitividad, Red Temática de Investigación Cooperativa en Cáncer (RD12/0036/0027 to PSG and AHL).

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