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

Synthesis of dry SmCl₃ from Sm₂O₃ revisited. Implications for the encapsulation of samarium compounds into carbon nanotubes

Samarium is a rare-earth metal with several applications in materials science. It is used in organic chemistry as a reducing agent and it is the active payload in samarium-153 lexidronam, a drug being used for palliative treatment of bone metastases. Recently, the encapsulation of samarium compounds into the cavities of carbon nanotubes has attracted interest for the development of the next generation of radiopharmaceuticals. In the present study, we explore different routes to afford the encapsulation of samarium based materials into single-walled carbon nanotubes. Anhydrous samarium(III) chloride, despite being highly hygroscopic, raises as an excellent candidate to achieve a high filling efficiency. We provide a protocol that allows the synthesis of anhydrous samarium(III) chloride starting from samarium(III) oxide in a fast and simple manner. Synchrotron X-ray powder diffraction confirmed the crystallinity and purity of the synthesized SmCl₃

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

Synthesis of dry SmCl₃ from Sm₂O₃ revisited. Implications for the encapsulation of samarium compounds into carbon nanotubes

Samarium is a rare-earth metal with several applications in materials science. It is used in organic chemistry as a reducing agent and it is the active payload in samarium-153 lexidronam, a drug being used for palliative treatment of bone metastases. Recently, the encapsulation of samarium compounds into the cavities of carbon nanotubes has attracted interest for the development of the next generation of radiopharmaceuticals. In the present study, we explore different routes to afford the encapsulation of samarium based materials into single-walled carbon nanotubes. Anhydrous samarium(III) chloride, despite being highly hygroscopic, raises as an excellent candidate to achieve a high filling efficiency. We provide a protocol that allows the synthesis of anhydrous samarium(III) chloride starting from samarium(III) oxide in a fast and simple manner. Synchrotron X-ray powder diffraction confirmed the crystallinity and purity of the synthesized SmCl₃

A multi-proxy Late-glacial palaeoenvironmental record from Lake Bled, Slovenia

This study investigates the palaeoecological record (δ18O, δ13C, pollen, plant macrofossils, chironomids and cladocera) at Lake Bled (Slovenia) sedimentary core to better understand the response of terrestrial and aquatic ecosystems to Late-glacial climatic fluctuations. The multi-proxy record suggests that in the Oldest Dryas, the landscape around Lake Bled was rather open, presumably because of the cold and dry climate, with a trend towards wetter conditions, as suggested by an increase in tree pollen as well as chironomid and cladocera faunas typical for well-oxygenated water. Climatic warming at the beginning of the Late-glacial Interstadial at ca. 14,800 cal yr BP is suggested by an increase in the δ18O value, the appearance of Betula and Larix pollen and macrofossils, and a warmth-adapted chironomid fauna. With further warming at ca. 13,800 cal yr BP, broad-leaved tree taxa (Quercus, Tilia, Ulmus), Artemisia, and Picea increase, whereas chironomid data (Cricotopus B) suggest lowering of lake levels. After 12,800 cal yr BP (and throughout the Younger Dryas), the climate was colder and drier, as indicated by lower δ18O values, decline of trees, increase of microscopic charcoal, xerophytes and littoral chironomids. A warmer climate, together with the spread of broad-leaved tree taxa and a deeper, more productive lake, mark the onset of the Late-glacial/Holocene transition. These results suggest that terrestrial and aquatic ecosystems at Lake Bled were very dynamic and sensitive to Late-glacial climatic fluctuations

Non-cytotoxic carbon nanocapsules synthesized via one-pot filling and end-closing of multi-walled carbon nanotubes

Filled carbon nanotubes (CNTs) find application in a variety of fields that expand from sensors to supercapacitors going through targeted therapies. Bulk filling of CNTs in general results in samples that contain a large amount of non-encapsulated material external to the CNTs. The presence of external material can dominate the properties of the resulting hybrids and can also induce side effects when employed in the biomedical field. Unless the encapsulated payloads have a strong interaction with the inner CNT walls, an additional step is required to block the ends of the CNTs thus allowing the selective removal of the non-encapsulated compounds while preserving the inner cargo. Herein we present a fast, easy and versatile approach that allows both filling (NaI, KI, BaI, GdCl and SmCl) and end-closing of multi-walled CNTs in a single-step, forming "carbon nanocapsules". Remarkably the encapsulation of GdCl and SmCl leads to the formation of tubular van der Waals heterostructures. The prepared nanocapsules are efficiently internalized by cells without inducing cytotoxicity, thus presenting a safe tool for the delivery of therapeutic and dianostic agents to cells. The synergies of novel carbon and inorganic hybrid materials can be explored using the present approach

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

Non-cytotoxic carbon nanocapsules synthesized via one-pot filling and end-closing of multi-walled carbon nanotubes

Filled carbon nanotubes (CNTs) find application in a variety of fields that expand from sensors to supercapacitors going through targeted therapies. Bulk filling of CNTs in general results in samples that contain a large amount of non-encapsulated material external to the CNTs. The presence of external material can dominate the properties of the resulting hybrids and can also induce side effects when employed in the biomedical field. Unless the encapsulated payloads have a strong interaction with the inner CNT walls, an additional step is required to block the ends of the CNTs thus allowing the selective removal of the non-encapsulated compounds while preserving the inner cargo. Herein we present a fast, easy and versatile approach that allows both filling (NaI, KI, BaI, GdCl and SmCl) and end-closing of multi-walled CNTs in a single-step, forming "carbon nanocapsules". Remarkably the encapsulation of GdCl and SmCl leads to the formation of tubular van der Waals heterostructures. The prepared nanocapsules are efficiently internalized by cells without inducing cytotoxicity, thus presenting a safe tool for the delivery of therapeutic and dianostic agents to cells. The synergies of novel carbon and inorganic hybrid materials can be explored using the present approach

A multi-proxy Late-glacial palaeoenvironmental record from Lake Bled, Slovenia

This study investigates the palaeoecological record (delta O-18, delta C-13, pollen, plant macrofossils, chironomids and cladocera) at Lake Bled (Slovenia) sedimentary core to better understand the response of terrestrial and aquatic ecosystems to Late-glacial climatic fluctuations. The multi-proxy record suggests that in the Oldest Dryas, the landscape around Lake Bled was rather open, presumably because of the cold and dry climate, with a trend towards wetter conditions, as suggested by an increase in tree pollen as well as chironomid and cladocera faunas typical for well-oxygenated water. Climatic warming at the beginning of the Late-glacial Interstadial at ca. 14,800 cal yr BP is suggested by an increase in the delta O-18 value, the appearance of Betula and Larix pollen and macrofossils, and a warmth-adapted chironomid fauna. With further warming at ca. 13,800 cal yr BP, broad-leaved tree taxa (Quercus, Tilia, Ulmus), Artemisia, and Picea increase, whereas chironomid data (Cricotopus B) suggest lowering of lake levels. After 12,800 cal yr BP (and throughout the Younger Dryas), the climate was colder and drier, as indicated by lower delta O-18 values, decline of trees, increase of microscopic charcoal, xerophytes and littoral chironomids. A warmer climate, together with the spread of broad-leaved tree taxa and a deeper, more productive lake, mark the onset of the Late-glacial/Holocene transition. These results suggest that terrestrial and aquatic ecosystems at Lake Bled were very dynamic and sensitive to Late-glacial climatic fluctuations.status: publishe