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-NH2 (1)] or samarium chloride-filled amino-functionalized CNTs with [SmCl3@SWCNT-mAb (3)] or without [SmCl3@SWCNT-NH2 (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.

Functionalised carbon nanotubes: From intracellular uptake and cell-related toxicity to systemic brain delivery.

Carbon nanotubes (CNTs) have long been regarded as promising carriers in biomedicine. Due to their high surface area and unique needle-like structure, CNTs are uniquely equipped to carry therapeutic molecules across biological membranes and, therefore, have been widely researched for use in theranostic applications. The attractive properties of the CNTs entice also their use in the brain environment. Cutting edge brain-specific therapies, capable of circumventing the physical and biochemical blockage of the blood-brain barrier, could be a precious tool to tackle brain disorders. With an increasing number of applications and expanding production, the effects of direct and indirect exposure to CNTs on cellular and molecular levels and more globally the general health, must be carefully assessed and limited. In this chapter, we review the most recent trends on the development and application of CNT-based nanotechnologies, with a particular focus on the carrier properties, cell internalisation and processing, and mechanisms involved in cell toxicity. Novel approaches for CNT-based systemic therapeutic brain delivery following intravenous administration are also reviewed. Moreover, we highlight fundamental questions that should be addressed in future research involving CNTs, aiming at achieving its safe introduction into the clinics.

The Shortening of MWNT-SPION Hybrids by Steam Treatment Improves Their Magnetic Resonance Imaging Properties In Vitro and In Vivo.

Carbon nanotubes (CNTs) have been advocated as promising nanocarriers in the biomedical field. Their high surface area and needle-like shape make these systems especially attractive for diagnostic and therapeutic applications. Biocompatibility, cell internalization, biodistribution, and pharmacokinetic profile have all been reported to be length dependent. In this study, further insights are gotten on the role that the length of CNTs plays when developing novel contrast agents for magnetic resonance imaging (MRI). Two samples of CNTs with different length distribution have been decorated with radio-labeled iron oxide nanoparticles. Despite characterization of the prepared hybrids reveals a similar degree of loading and size of the nanoparticles for both samples, the use of short CNTs is found to enhance the MRI properties of the developed contrast agents both in vitro and in vivo compared to their long counterparts.

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.

Kupffer Cell Isolation for Nanoparticle Toxicity Testing.

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle toxicity testing in immortalized cell lines or primary cells have shown discrepancies, highlighting the need to extend the use of primary cells for in vitro assays. This protocol describes the isolation of mouse liver macrophages, named Kupffer cells, and their use to study nanoparticle toxicity. Kupffer cells are the most abundant macrophage population in the body and constitute part of the reticulo-endothelial system (RES), responsible for the capture of circulating nanoparticles. The Kupffer cell isolation method reported here is based on a 2-step perfusion method followed by purification on density gradient. The method, based on collagenase digestion and density centrifugation, is adapted from the original protocol developed by Smedsrød et al. designed for rat liver cell isolation and provides high yield (up to 14 x 10(6) cells per mouse) and high purity (>95%) of Kupffer cells. This isolation method does not require sophisticated or expensive equipment and therefore represents an ideal compromise between complexity and cell yield. The use of heavier mice (35-45 g) improves the yield of the isolation method but also facilitates remarkably the procedure of portal vein cannulation. The toxicity of functionalized carbon nanotubes f-CNTs was measured in this model by the modified LDH assay. This method assesses cell viability by measuring the lack of structural integrity of Kupffer cell membrane after incubation with f-CNTs. Toxicity induced by f-CNTs can be measured consistently using this assay, highlighting that isolated Kupffer cells are useful for nanoparticle toxicity testing. The overall understanding of nanotoxicology could benefit from such models, making the nanoparticle selection for clinical translation more efficient.

Polyethylene glycol conjugated polymeric nanocapsules for targeted delivery of quercetin to folate-expressing cancer cells in vitro and in vivo.

In this work we describe the formulation and characterization of chemically modified polymeric nanocapsules incorporating the anticancer drug, quercetin, for the passive and active targeting to tumors. Folic acid was conjugated to poly(lactide-co-glycolide) (PLGA) polymer to facilitate active targeting to cancer cells. Two different methods for the conjugation of PLGA to folic acid were employed utilizing polyethylene glycol (PEG) as a spacer. Characterization of the conjugates was performed using FTIR and (1)H NMR studies. The PEG and folic acid content was independent of the conjugation methodology employed. PEGylation has shown to reduce the size of the nanocapsule; moreover, zeta-potential was shown to be polymer-type dependent. Comparative studies on the cytotoxicity and cellular uptake of the different formulations by HeLa cells, in the presence and absence of excess folic acid, were carried out using MTT assay and Confocal Laser Scanning Microscopy, respectively. Both results confirmed the selective uptake and cytotoxicity of the folic acid targeted nanocapsules to the folate enriched cancer cells in a folate-dependent manner. Finally, the passive tumor accumulation and the active targeting of the nanocapsules to folate-expressing cells were confirmed upon intravenous administration in HeLa or IGROV-1 tumor-bearing mice. The developed nanocapsules provide a system for targeted delivery of a range of hydrophobic anticancer drugs in vivo.

Magnetically Decorated Multi-Walled Carbon Nanotubes as Dual MRI and SPECT Contrast Agents.

Carbon nanotubes (CNTs) have been proposed as one of the most promising nanomaterials to be used in biomedicine for their applications in drug/gene delivery as well as biomedical imaging. The present study developed radio-labeled iron oxide decorated multi-walled CNTs (MWNT) as dual magnetic resonance (MR) and single photon emission computed tomography (SPECT) imaging agents. Hybrids containing different amounts of iron oxide were synthesized by in situ generation. Physicochemical characterisations revealed the presence of superparamagnetic iron oxide nanoparticles (SPION) granted the magnetic properties of the hybrids. Further comprehensive examinations including high resolution transmission electron microscopy (HRTEM), fast Fourier transform simulations (FFT), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) assured the conformation of prepared SPION as γ-Fe2O3. High r2 relaxivities were obtained in both phantom and in vivo MRI compared to the clinically approved SPION Endorem®. The hybrids were successfully radio-labeled with technetium-99m through a functionalized bisphosphonate and enabled SPECT/CT imaging and γ-scintigraphy to quantitatively analyze the biodistribution in mice. No abnormality was found by histological examination and the presence of SPION and MWNT were identified by Perls stain and Neutral Red stain, respectively. TEM images of liver and spleen tissues showed the co-localization of SPION and MWNT within the same intracellular vesicles, indicating the in vivo stability of the hybrids after intravenous injection. The results demonstrated the capability of the present SPION-MWNT hybrids as dual MRI and SPECT contrast agents for in vivo use.

Intraoperative fluorescence imaging of peritoneal dissemination of ovarian carcinomas. A preclinical study.

OBJECTIVE: Improvement of the management and outcome of ovarian cancers may require intraoperative detection and therapeutic intervention to treat minimal residual disease after complete surgery. The aim of this study was to validate the importance of fluorescence in the peroperative detection of human ovarian adenocarcinoma cells and to determine its efficiency in detecting infra millimetric tumor metastases. METHODS: A fluorescent RAFT-(cRGD)4 tracer molecule (AngioStamp®) was used. The tracer is based on a biomarker, which has a very high affinity for the α(v)ß3 integrin, which is overexpressed in a large ratio of cancer cells and neovessel endothelial cells during angiogenesis. Infrared fluorescence was visualized with Fluobeam®, an open fluorescent imaging system that could potentially be used in peroperative conditions in the future. RESULTS: This novel technique allowed the specific detection of residual tumor deposits and inframillimetric metastases, smaller than 500µm, which were resected under fluorescent guidance. AngioStamp® was able to detect all types of cell lines, derived from human ovarian adenocarcinomas, before or after chemotherapy treatment in animals. The effectiveness of AngioStamp® for the detection of various human ovarian adenocarcinomas was assessed on 10 different fragments of tumor, implanted subcutaneously in nude mice. All implanted tumor fragments were visualized by AngioStamp®. CONCLUSIONS: The high rate of recurrence after apparently complete surgery and/or complete clinical response to chemotherapy implies that most patients have undetected minimal residual disease. Novel techniques such as laparoscopic or laparotomic fluorescence may prove to be crucial in reassessing the definition of primary outcome in ovarian cancer management.