Évaluation et caractérisation de l'efficacité thérapeutique de nanoparticules multifonctionnelles pour de la thérapie photodynamique interstitielle appliquée au glioblastome

Despite heavy treatments, local recurrence of glioblastoma (GBM) remains frequent, leading to a poor prognosis with a median survival of 15 months. One of the main challenges is to eradicate the infiltrating part of the tumor tissue that causes recurrence while preserving the healthy brain parenchyma from irreversible damage. To this end, neuropilin-1 (NRP-1), a receptor overexpressed by endothelial cells of angiogenic phenotype of the tumor vasculature and associated with proliferation and migration of GBM has been demonstrated to be a relevant molecular target to promote the anti-vascular effect of photodynamic therapy (PDT). The KDKPPR peptide moiety targeting NRP-1 and a porphyrin acting as a photosensitizer (PS) have thus been grafted onto AGuIX design nanoparticles to meet these challenges. The photophysical properties of the PS and the molecular affinity of the KDKPPR peptide for the NRP-1 recombinant protein were preserved after the functionalization of the AGuIX nanoparticle. In vivo selectivity studies revealed prolonged retention of peptide-conjugated nanoparticles in the vascular system of xenografted human GBMs. Post-PDT follow-ups have shown promising responses associating delayed tumor growth and a reduction in tumor metabolism in the days following treatment. We also demonstrated an acute inflammation and the formation of edema within and around the tumor in the days following the treatment as well as an influx of macrophages in the tumor which in these conditions could largely participate in the inflammatory and post-PDT immunogen response. Finally, by using a monocytic cell line (THP-1) brought into contact with the conditioned medium from tumor cells treated with PDT, we were able to demonstrate the immunostimulatory effects of PDT and in particular its potential to induce the polarization of macrophages towards the inflammatory and anti-tumor phenotype M1.Malgré des traitements lourds, les récidives locales du glioblastome (GBM) restent fréquentes, conduisant à un pronostic sombre avec une médiane de survie de 15 mois. L’un des principaux défis à relever est d’éradiquer la partie infiltrante du tissu tumoral à l’origine des récidives tout en préservant le parenchyme cérébral sain de lésions irréversibles. Dans ce but, la neuropiline-1 (NRP-1), un récepteur surexprimé par les cellules endothéliales de phénotype angiogénique du système vasculaire tumoral et associé à la prolifération et à la migration du GBM a été mise en évidence comme étant une cible moléculaire pertinente pour promouvoir l'effet anti-vasculaire de la thérapie photodynamique (PDT). Le peptide KDKPPR ciblant NRP-1 et une porphyrine jouant le rôle de photosensibilisateur (PS) ont ainsi été greffés sur des nanoparticules de design AGuIX pour répondre à ces enjeux. Les propriétés photophysiques du PS et l'affinité moléculaire du peptide KDKPPR pour la protéine recombinante NRP-1 ont été préservées après la fonctionnalisation de la nanoparticule AGuIX. Les études de sélectivité in vivo ont révélé une rétention prolongée des nanoparticules conjuguées aux peptides dans le système vasculaire des GBM humains xénogreffés. Les suivis post-PDT ont démontré des réponses prometteuses associant un retard de croissance tumorale et une chute du métabolisme tumoral dans les jours suivant le traitement. Nous avons également mis en évidence une inflammation aiguë et la formation d’œdèmes au sein et au pourtour de la tumeur dans les jours suivant le traitement ainsi qu’une affluence de macrophages dans la tumeur qui dans ces conditions pourrait largement participer à la réponse inflammatoire et immunogène post-PDT. Finalement, en utilisant une lignée cellulaire monocytaire (THP-1) mis au contact avec le milieu conditionné provenant de cellules tumorales traitées par PDT, nous avons pu démontrer les effets immunostimulants de la PDT et notamment son potentiel à induire la polarisation des macrophages vers le phénotype inflammatoire et antitumoral M1

Gadolinium-based nanoparticles for the treatment of glioblastoma by MRI-guided photodynamic therapy

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Ultrasmall AGuIX theranostic nanoparticles for vascular-targeted interstitial photodynamic therapy of glioblastoma

International audienceDespite combined treatments, glioblastoma outcome remains poor with frequent local recurrences, indicating that a more efficient and local therapy is needed. In this way, vascular-targeted photodynamic therapy (VTP) is a promising complementary approach to improve tumor eradication1 by destroying its neovessels2. In this study, we designed a polysiloxane-based nanoparticle (NP) combining a magnetic resonance imaging (MRI) contrast agent, a photosensitizer (PS) and a new ligand peptide motif (KDKPPR) targeting neuropilin-1 (NRP-1)3,4, a receptor overexpressed by angiogenic endothelial cells of the tumor vasculature. This structure achieves the detection of the tumor tissue and its proliferating part by MRI analysis, followed by its treatment by VTP. The photophysical properties of the PS and the peptide affinity for NRP-1 recombinant protein were preserved after the functionalization of NPs. Cellular uptake of NPs by human umbilical vein endothelial cells (HUVEC) was increased twice compared to NPs without the KDKPPR peptide moiety or conjugated with a scramble peptide. NPs induced no cytotoxicity without light exposure but conferred a photocytotoxic effect to cells after photodynamic therapy (PDT)5. In vivo, complementary approaches were investigated, combining non-invasive imaging by magnetic resonance or by fluorescence and a tissue assay by ICP-MS to determine gadolinium concentrations, revealing renal and hepatic eliminations. The tumor selectivity of the NPs compared to the healthy brain parenchyma was validated by MRI data. The in vivo selectivity, evaluated using a skinfold chamber model in mice, confirms that the functionalized NPs with KDKPPR peptide moiety were localized in the tumor vessel wall, validating this vascular targeting strategy

Ultrasmall AGuIX theranostic nanoparticles for vascular-targeted interstitial photodynamic therapy of glioblastoma

International audienceDespite combined treatments, glioblastoma outcome remains poor with frequent local recurrences, indicating that a more efficient and local therapy is needed. In this way, vascular-targeted photodynamic therapy (VTP) could help tumor eradication by destroying its neovessels. In this study, we designed a polysiloxane-based nanoparticle (NP) combining a magnetic resonance imaging (MRI) contrast agent, a photosensitizer (PS) and a new ligand peptide motif (KDKPPR) targeting neuropilin-1 (NRP-1), a receptor overexpressed by angiogenic endothelial cells of the tumor vasculature. This structure achieves the detection of the tumor tissue and its proliferating part by MRI analysis, followed by its treatment by VTP. The photophysical properties of the PS and the peptide affinity for NRP-1 recombinant protein were preserved after the functionalization of NPs. Cellular uptake of NPs by human umbilical vein endothelial cells (HUVEC) was increased twice compared to NPs without the KDKPPR peptide moiety or conjugated with a scramble peptide. NPs induced no cytotoxicity without light exposure but conferred a photocytotoxic effect to cells after photodynamic therapy (PDT). The in vivo selectivity, evaluated using a skinfold chamber model in mice, confirms that the functionalized NPs with KDKPPR peptide moiety were localized in the tumor vessel wall

AGUIX® theranostic nanoparticles for vascular-targeted interstitial photodynamic therapy of brain tumors

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Targeting Glioblastoma-Associated Macrophages for Photodynamic Therapy Using AGuIX®-Design Nanoparticles

Glioblastoma (GBM) is the most difficult brain cancer to treat, and photodynamic therapy (PDT) is emerging as a complementary approach to improve tumor eradication. Neuropilin-1 (NRP-1) protein expression plays a critical role in GBM progression and immune response. Moreover, various clinical databases highlight a relationship between NRP-1 and M2 macrophage infiltration. In order to induce a photodynamic effect, multifunctional AGuIX®-design nanoparticles were used in combination with a magnetic resonance imaging (MRI) contrast agent, as well as a porphyrin as the photosensitizer molecule and KDKPPR peptide ligand for targeting the NRP-1 receptor. The main objective of this study was to characterize the impact of macrophage NRP-1 protein expression on the uptake of functionalized AGuIX®-design nanoparticles in vitro and to describe the influence of GBM cell secretome post-PDT on the polarization of macrophages into M1 or M2 phenotypes. By using THP-1 human monocytes, successful polarization into the macrophage phenotypes was argued via specific morphological traits, discriminant nucleocytoplasmic ratio values, and different adhesion abilities based on real-time cell impedance measurements. In addition, macrophage polarization was confirmed via the transcript-level expression of TNFα, CXCL10, CD-80, CD-163, CD-206, and CCL22 markers. In relation to NRP-1 protein over-expression, we demonstrated a three-fold increase in functionalized nanoparticle uptake for the M2 macrophages compared to the M1 phenotype. The secretome of the post-PDT GBM cells led to nearly a three-fold increase in the over-expression of TNFα transcripts, confirming the polarization to the M1 phenotype. The in vivo relationship between post-PDT efficiency and the inflammatory effects points to the extensive involvement of macrophages in the tumor zone.Title in Web of Science: Targeting Glioblastoma-Associated Macrophages for Photodynamic Therapy Using AGuIX((R))-Design Nanoparticles</p

Multiscale selectivity and in vivo biodistribution of NRP-1-targeted theranostic AGuIX nanoparticles for PDT of glioblastoma

International audienceBackground: Local recurrences of glioblastoma (GBM) after heavy standard treatments remain frequent and lead to a poor prognostic. Major challenges are the infiltrative part of the tumor tissue which is the ultimate cause of recurrence. The therapeutic arsenal faces the difficulty of eradicating this infiltrating part of the tumor tissue while increasing the targeting of tumor and endogenous stromal cells such as angiogenic endothelial cells. In this aim, neuropilin-1 (NRP-1), a transmembrane receptor mainly overexpressed by endothelial cells of the tumor vascular system and associated with malignancy, proliferation and migration of GBM, highlighted to be a relevant molecular target to promote the anti-vascular effect of photodynamic therapy (VTP).Methods: The multiscale selectivity was investigated for KDKPPR peptide moiety targeting NRP-1 and a porphyrin molecule as photosensitizer (PS), both grafted onto original AGuIX design nanoparticle. AGuIX nanoparticle, currently in Phase II clinical trials for the treatment of brain metastases with radiotherapy, allows to achieve a real-time magnetic resonance imaging (MRI) and an accumulation in the tumor area by EPR (enhanced permeability and retention) effect. Using surface-plasmon resonance (SPR), we evaluated the affinities of KDKPPR and scramble free peptides, and also peptides-conjugated AGuIX nanoparticles to recombinant rat and human NRP-1 proteins. For in vivo selectivity, we used a cranial window model and parametric maps obtained from T2*-weighted perfusion MRI analysis.Results: The photophysical characteristics of the PS and KDKPPR molecular affinity for recombinant human NRP-1 proteins were maintained after the functionalization of AGuIX nanoparticle with a dissociation constant of 4.7 μM determined by SPR assays. Cranial window model and parametric maps, both revealed a prolonged retention in the vascular system of human xenotransplanted GBM. Thanks to the fluorescence of porphyrin by non-invasive imaging and the concentration of gadolinium evaluated after extraction of organs, we checked the absence of nanoparticle in the brains of tumor-free animals and highlighted elimination by renal excretion and hepatic metabolism.Conclusion: Post-VTP follow-ups demonstrated promising tumor responses with a prolonged delay in tumor growth accompanied by a decrease in tumor metabolism