Ultrasound-triggered repolarization of microglia cells using piezoelectric nanoparticles for enhanced immunotherapy against glioblastoma multiforme

Glioblastoma multiforme (GBM) is one of the most aggressive types of primary brain tumour. It is characterized by a rapid and drug-resistant progression, high morbidity, and mortality. To survive, GBM cells constitute a genetically heterogenous tumour-microenvironment (TME), recruiting surrounding healthy brain cells by releasing various intercellular signals. Among these, associated microglia (GAMs) represent the largest population. Once engaged in the TME, microglia acquire a specific activation of the anti-inflammatory and pro-tumour M2 phenotype. On the other hand, “classically activated” microglia usually express the opposite phenotype (M1), with pro-inflammatory and antitumorigenic activity, that is expected to exert a beneficial effect in glioblastoma treatment. In this work, we developed an innovative targeted anti-glioma immunotherapy, based on pro-inflammatory modulation of GAMs phenotype, through a controlled and localized electrical impulse. The proposed strategy relies on the excitation of 𝛽- poly (vinylidene fluoride-trifluoro ethylene) (𝛽 -P(VDF-TrFE)) piezoelectric nanoparticles (PNPs) through remote ultrasound application (US). Homotypic targeting is achieved by coating the PNPs with U87-MG GBM cell membrane extract (to obtain CM-PNPs). Such camouflaged piezoelectric nanovectors locally generate electrical cues on GAM membranes upon ultrasound (US) stimulation, ultimately triggering their selective expression and secretion of pro-inflammatory chemoattractant factors. Piezoelectric features of our nanoplatform were characterized by a new non-contact piezoelectric force microscopy (PFM) modality, for improving the accuracy of the evaluation of the converse piezoelectric coefficient (d33) of soft nanostructured materials. US stimulation was modelled through a numerical finite element software, indicating that a US pressure of 0.1 MPa can reach the cells and activate nanoparticle piezoelectricity. As a hint of M1 polarization of microglia after treatment, expression of membrane inflammatory receptors CD40, CD86, and CD206 showed an increased expression level. Investigation of calcium (Ca2+) signalling shows a significant increase of Ca2+ influx through voltage-gated channels in treated microglia compared to non-treated groups, suggesting that the CAMKK2-NF-𝜅B pathway could be established to induce M1-like polarization. These preliminary activation results led us to push the investigation further. Thus, we assessed microglia activation under U87-MG-conditioned medium, to evaluate the efficacy of CM-PNPs+US treatment in repolarizing microglia from M2 to M1 phenotype. Markers of M1 activation showed an increase in expression in the CM-PNPs+US treated group. However, it was lower than in the unconditioned experiment, as we expected from the immunosuppressive action of U87-MG-released cytokines in the medium. A Transwell co-culture experiment assessed the beneficial anti-tumour activity of piezo-activated microglia. Confocal imaging revealed a significantly lower fluorescence intensity of the Ki-67 tumour proliferation marker in the sample co-cultured with treated microglia. Moreover, when treated with piezo-activated microglia cells, U87-MG cells show a decrement in metabolic activity. These results pave the way for further investigation in a more biomimetic TME, employing, for instance, an organoids model, and a microfluidic device that resembles the blood-brain barrier, to evaluate the dynamic targeting capability of such a smart bio nanosystem.