Design of antibody-functionalized carbon nanotubes filled with radioactivable metals towards a targeted anticancer therapy

Spinato, Cinzia et al.In the present work we have devised the synthesis of a novel promising carbon nanotube carrier for the targeted delivery of radioactivity, through a combination of endohedral and exohedral functionalization. Steam-purified single-walled carbon nanotubes (SWCNTs) have been initially filled with radioactive analogues (i.e. metal halides) and sealed by high temperature treatment, affording closed-ended CNTs with the filling material confined in the inner cavity. The external functionalization of these filled CNTs was then achieved by nitrene cycloaddition and followed by the derivatization with a monoclonal antibody (Cetuximab) targeting the epidermal growth factor receptor (EGFR), overexpressed by several cancer cells. The targeting efficiency of the so-obtained conjugate was evaluated by immunostaining with a secondary antibody and by incubation of the CNTs with EGFR positive cells (U87-EGFR+), followed by flow cytometry, confocal microscopy or elemental analyses. We demonstrated that our filled and functionalized CNTs can internalize more efficiently in EGFR positive cancer cells.The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007–2013/ under REA grant agreement no 290023 (RADDEL). This work was partly supported by the Centre National de la Recherche Scientifique (CNRS), by the Agence Nationale de la Recherche (ANR) through the LabEx project Chemistry of Complex Systems (ANR-10-LABX-0026_CSC) and by the International Center for Frontier Research in Chemistry (icFRC). ICN2 acknowledges support from the Severo Ochoa Program (MINECO, Grant SEV-2013-0295). KCL acknowledges support from WCR. The authors are grateful to Thomas Swan & Co. Ltd for providing Elicarb® SWCNTs. MM, MK and EP work has been done as a part of PhD program in Materials Sciences at UAB.Peer reviewe

Design of antibody-functionalized carbon nanotubes filled with radioactivable metals towards a targeted anticancer therapy

In the present work we have devised the synthesis of a novel promising carbon nanotube carrier for the targeted delivery of radioactivity, through a combination of endohedral and exohedral functionalization. Steam-purified single-walled carbon nanotubes (SWCNTs) have been initially filled with radioactive analogues (i.e. metal halides) and sealed by high temperature treatment, affording closed-ended CNTs with the filling material confined in the inner cavity. The external functionalization of these filled CNTs was then achieved by nitrene cycloaddition and followed by the derivatization with a monoclonal antibody (Cetuximab) targeting the epidermal growth factor receptor (EGFR), overexpressed by several cancer cells. The targeting efficiency of the so-obtained conjugate was evaluated by immunostaining with a secondary antibody and by incubation of the CNTs with EGFR positive cells (U87-EGFR+), followed by flow cytometry, confocal microscopy or elemental analyses. We demonstrated that our filled and functionalized CNTs can internalize more efficiently in EGFR positive cancer cells

Développement de stratégies chimiques pour préparer des nanotubes multifonctionnels pour la thérapie anticancéreuse

The application of carbon nanotubes (CNTs) in the biomedical field has been widely explored thanks to their physico-chemical properties and their biocompatibility. By the external and/or internal functionalization of CNTs it is possible to prepare novel conjugates tailoring different properties and applications. We have investigated the covalent derivatization of CNTs by different chemical strategies to achieve suitable carriers for anticancer therapy. In one project, we have explored the conversion of the carboxylic groups of oxidized CNTs into amino groups, and the ability of these conjugates to complex genetic material, for gene delivery. In another project, CNTs have been functionalized with linkers bearing a cleavable disulfide bond, and further conjugated to a therapeutic nanobody for controlled intracellular drug release. Finally, we have investigated the reactivity of close-ended CNTs filled with radioactivable material toward Bingel and nitrene cycloadditions and the conjugation of a targeting antibody, for the target delivery of radioactivity. By several characterization techniques we have proved that the antibody is covalently grafted to the CNT-carrier and it still possesses its targeting ability. Investigations on the biological profile of these conjugates (cytotoxicity, targeting, uptake, biodistribution) have been also carried out.L’application de nanotubes de carbone (CNTs) dans le domaine biomédical a été largement explorée grâce à leur propriétés physico-chimiques et à leur biocompatibilité. Par la fonctionnalisation extérieur et/ou intérieur des CNTs c’est possible de préparer des nouveaux conjugués avec différentes propriétés et applications. On a exploré la modification des nanotubes par voie covalente pour leur utilisation comme vecteurs de biomolécules pour achever la thérapie anticancéreuse. Pendant ma thèse, j’ai travaillé sur trois projets: l’application de différentes approches pour la conversion des groupes acides carboxyliques de MWCNTs oxydés en amines, dans le but de préparer des conjugués capables de complexer du siRNA (petits ARN interférents). Dans un second projet, j’ai développé des conjugués à base de nanotubes de carbone couplés avec un fragment d’anticorps thérapeutique via une liaison clivable afin d’en étudier le potentiel antitumoral. Dans le dernier projet, on a achevé la fonctionnalisation de CNTs remplis avec des molécules radioactivables par cycloaddition de nitrene et ensuite conjugué un anticorps de ciblage tumoral. Le but été d’utiliser les nanotubes comme vecteurs pour la délivrance de radioactivité à l'intérieur des cellules tumorales ciblées par l’anticorps. On a aussi conduit des investigations biologique, afin d’évaluer la toxicité et l’efficace de ce conjugué

Different chemical strategies to aminate oxidised multi-walled carbon nanotubes for siRNA complexation and delivery

The carboxylic groups of oxidised multi-walled carbon nanotubes were directly converted into amino functions without extending the lateral chain. These nanotubes have been investigated as carriers for siRNA delivery.</p

Evaluation of the immunological profile of antibody-functionalized metal-filled single-walled carbon nanocapsules for targeted radiotherapy

This study investigates the immune responses induced by metal-filled single-walled carbon nanotubes (SWCNT) under in vitro, ex vivo and in vivo settings. Either empty amino-functionalized CNTs [SWCNT-NH(2) (1)] or samarium chloride-filled amino-functionalized CNTs with [SmCl(3)@SWCNT-mAb (3)] or without [SmCl(3)@SWCNT-NH(2) (2)] Cetuximab functionalization were tested. Conjugates were added to RAW 264.7 or PBMC cells in a range of 1 μg/ml to 100 μg/ml for 24 h. Cell viability and IL-6/TNFα production were determined by flow cytometry and ELISA. Additionally, the effect of SWCNTs on the number of T lymphocytes, B lymphocytes and monocytes within the PBMC subpopulations was evaluated by immunostaining and flow cytometry. The effect on monocyte number in living mice was assessed after tail vein injection (150 μg of each conjugate per mouse) at 1, 7 and 13 days post-injection. Overall, our study showed that all the conjugates had no significant effect on cell viability of RAW 264.7 but conjugates 1 and 3 led to a slight increase in IL-6/TNFα. All the conjugates resulted in significant reduction in monocyte/macrophage cell numbers within PBMCs in a dose-dependent manner. Interestingly, monocyte depletion was not observed in vivo, suggesting their suitability for future testing in the field of targeted radiotherapy in mice

Design of antibody-functionalized carbon nanotubes filled with radioactivable metals towards a targeted anticancer therapy

In the present work we have devised the synthesis of a novel promising carbon nanotube carrier for the targeted delivery of radioactivity, through a combination of endohedral and exohedral functionalization. Steam-purified single-walled carbon nanotubes (SWCNTs) have been initially filled with radioactive analogues (i.e. metal halides) and sealed by high temperature treatment, affording closed-ended CNTs with the filling material confined in the inner cavity. The external functionalization of these filled CNTs was then achieved by nitrene cycloaddition and followed by the derivatization with a monoclonal antibody (Cetuximab) targeting the epidermal growth factor receptor (EGFR), overexpressed by several cancer cells. The targeting efficiency of the so-obtained conjugate was evaluated by immunostaining with a secondary antibody and by incubation of the CNTs with EGFR positive cells (U87-EGFR+), followed by flow cytometry, confocal microscopy or elemental analyses. We demonstrated that our filled and functionalized CNTs can internalize more efficiently in EGFR positive cancer cells

Neutron-irradiated antibody-functionalised carbon nanocapsules for targeted cancer radiotherapy

Radiotherapy is a cancer treatment utilising high doses of ionizing radiation to destroy cancer cells. Our team has pioneered neutron activation of Sm, filled and sealed into single-walled (SWCNTs) and multi-walled carbon nanotubes (MWCNTs), to create stable and high-dose radioactive carbon nanocapsules for cancer radiotherapy. In this work, MWCNTs filled with enriched SmCl (Sm@MWCNTs) were sealed and irradiated, followed by surface functionalisation with an epidermal growth factor receptor (EGFR)-targeting antibody. Characterisation of functionalised Sm@MWCNTs was carried out using thermogravimetric analysis, gel electrophoresis and transmission electron microscopy. The organ biodistribution of the radioactive functionalised Sm@MWCNTs and therapeutic efficacy were studied in an experimental melanoma lung metastatic tumour model in mice after intravenous injection. Quantitative biodistribution analyses showed high accumulation of Sm@MWCNT-Ab in lung. Significant tumour growth reduction was induced by both treatments of Sm@MWCNTs functionalised with or without the antibody after a single intravenous injection. Although EGFR targeting showed no improvement in therapeutic efficacy, reduced spleen toxicity and normal haematological profiles were obtained for both functionalised derivatives. The current study demonstrated the possibility of performing chemical functionalisation and antibody conjugation on radioactive nanocapsules post-irradiation for the preparation of targeted radiopharmaceuticals

Neutron-irradiated antibody-functionalised carbon nanocapsules for targeted cancer radiotherapy

Radiotherapy is a cancer treatment utilising high doses of ionizing radiation to destroy cancer cells. Our team has pioneered neutron activation of 152Sm, filled and sealed into single-walled (SWCNTs) and multi-walled carbon nanotubes (MWCNTs), to create stable and high-dose radioactive carbon nanocapsules for cancer radiotherapy. In this work, MWCNTs filled with enriched 152SmCl3 (Sm@MWCNTs) were sealed and irradiated, followed by surface functionalisation with an epidermal growth factor receptor (EGFR)-targeting antibody. Characterisation of functionalised Sm@MWCNTs was carried out using thermogravimetric analysis, gel electrophoresis and transmission electron microscopy. The organ biodistribution of the radioactive functionalised 153Sm@MWCNTs and therapeutic efficacy were studied in an experimental melanoma lung metastatic tumour model in mice after intravenous injection. Quantitative biodistribution analyses showed high accumulation of 153Sm@MWCNT-Ab in lung. Significant tumour growth reduction was induced by both treatments of 153Sm@MWCNTs functionalised with or without the antibody after a single intravenous injection. Although EGFR targeting showed no improvement in therapeutic efficacy, reduced spleen toxicity and normal haematological profiles were obtained for both functionalised derivatives. The current study demonstrated the possibility of performing chemical functionalisation and antibody conjugation on radioactive nanocapsules post-irradiation for the preparation of targeted radiopharmaceuticals.This work received funding from the European Union’s Seventh Framework Programme (FP7-ITN Marie-Curie Actions, RADDEL, 290023). PM Costa would like to acknowledge the funding from the Wellcome Trust (WT103913). KT A-J would like to acknowledge the funding from Worldwide Cancer Research, UK (12–1054). This work was partly supported by the Centre National de la Recherche Scientifique (CNRS), Agence Nationale de la Recherche (ANR) through the LabEx project Chemistry of Complex Systems (ANR-10-LABX-0026_CSC), and the International Center for Frontier Research in Chemistry (icFRC). ICMAB and ICN2 acknowledge financial support from the Spanish Ministry of Economy and Competitiveness, through the “Severo Ochoa” Programme for Centres of Excellence in R&D (SEV-2015-0496 and SEV-2017-0706). We thank Thomas Swan Co. Ltd. for supplying CNT Elicarb® samples. We wish to acknowledge Cathy Royer and Valérie Demais for TEM analyses at the Plateforme Imagerie in Vitro at the Center of Neurochemistry (Strasbourg, France).Peer reviewe

The secretory senescence in otorhinolaryngology: principles of treatment

Atrophy or hypofunction of the salivary gland because of aging, radio-therapy or disease causes hyposalivation and impairs the quality of life of patients by compromising mastication, swallowing and speech and by leading to a loss of taste. Moreover, hyposalivation exacerbates dental caries and induces periodontal disease, and oral candidiasis. Currently, no satisfactory therapies have been established to solve salivary hypofunction. Current treatment options for atrophy or hypofunction of the salivary glands in clinical practice are only symptomatic and include saliva substitutes and parasympathetic agonists, such as pilocarpine, to stimulate salivary flow. However, parasympathomimetics have systemic side effects, so different treatment options are necessary, and research has recently focused on this. The main strategies that have been proposed to restore salivary gland atrophy and hypofunction are gene therapy by gene activation/silencing during stem cell differentiation and by the use of viral vectors, such as adenoviruses; cell-based therapy with salivary gland cells, stem cells and non-salivary gland and/or non-epithelial cells to regenerate damaged salivary gland cells; replacement with tissue bioengineering in which organoids from pluripotent stem cells are used in the development of organ replacement regenerative therapy. Remarkable progression in this research field has been made in the last decade, but a definitive therapy for salivary gland hypofunction has not been developed due to intrinsic challenges that come with each approach. However, with research efforts in the future, a range of precision medicine therapies may become available individualized to each patient