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GNTs to safely label porcine bone marrow-derived mesenchymal stem cells that displayed excellent image contrast in phantom MRI. Our group reported an interesting and original application of SWCNTs to induce anisotropy in the di usion of water molecules in a phantom [103]. This work o ers new perspectives for contrast generation in di usion tensor magnetic resonance imaging, a powerful MRI technique to explore the microstructure of healthy and pathological brain.

The near-infrared radiation (NIR) absorption property of CNTs [152] can also be used to image their biodistribution in vivo. Yudasaka et al. [153] covered SWCNTs with a biocompatible polymer, which accumulated in brown fat, providing an imaging tool to visualize the distribution of this tissue in a preclinical model. Kim et al. [154] developed SWCNTs coated with gold and conjugated with antibody specific to the lymphatic vessel endothelial hyaluronan receptor to image the lymphatic vessels in mice. They induced a temperature increase of CNTs by NIR absorption using a laser beam and detected the nanotubes in the lymphatic vessels using photoacoustic and photothermal imaging. The NIR imaging techniques can also be combined with NIR guide photothermal therapy [155]. In this work, Liang et al. functionalized SWCNTs with polyethylene glycol to administrate them in BALB/c mice carrying 4T1 murine breast tumors in the inner knee. CNTs were directly injected on the primary tumor and visualized using photothermal imaging and MRI, not only in the tumor but also in the nearest metastatic lymph.

CNTs can be functionalized with di erent radioisotopes like Y-86 [156], C-14 [157], I-125 [158], Tc-99 m [159] or Cu-64 [160], making them promising CAs for nuclear medicine approaches. Al-Jamal and coworkers [161] used single-photon emission computed tomography (SPECT)/computed tomography (CT) imaging to study in vivo the internalization of three di erent MWCNTs radiolabeled with 111In (Fig. 9).

Furthermore, the particular features of carbon nanotubes make them good candidates to act as multimodal contrast agents with di erent imaging techniques.

4.2 Therapeutic Applications

Nanoparticles have revolutionized the field of drug delivery in the last two decades. Currently used therapeutic drugs su er from low selectivity and low half-life, making necessary it to give high doses to achieve the expected response, and thus increasing undesirable side-e ects. A proper and selective drug delivery system would overcome most of these issues, and carbon nanotubes have been widely studied for this end [15].

As previously indicated, the nature of nanotubes permits a versatile chemistry allowing the attachment of drugs in a covalent or non-covalent manner with an e cient drug-loading capacity. Besides, these nanostructures can be appropriately functionalized with di erent hydrophilic molecules to specifically recognize the receptors overexpressed in the target cells, according to the alterations related to the specific pathology to be treated [162]. So, the optimal functionalization of CNTs make them promising drug-delivery systems in numerous therapies owing to their achieved biocompatibility, suitable size, ability to penetrate the cells, and

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Fig. 9  Nano-SPECT/CT fused images of the whole body of a mouse. Images were recorded at 0.5, 3.5, and 24 h after injection of the 111In-DTPA-MWCNTs structures shown on the left side (with permission from Al-Jamal et al. [161])

their exceptional cell transfection capabilities [163, 164]. Hopeful studies have been reported to treat some of the most devastating pathologies nowadays, like cancer and neurodegenerative diseases, with CNT-based therapies using them either as drug carrier and delivery systems or taking advantage of nanotube physicochemical properties.

4.2.1 Cancer Treatment

Nowadays, the main challenge of cancer therapies relies on the preparation of smart carriers able to accurately target the tumoral cells and to perform a controlled release of the therapeutic drug in response to the tumor microenvironment properties. The classic approach of nanotubes for cancer treatment is based on the transport and delivery of chemotherapeutic drugs that su ers from systemic toxicity, narrow therapeutic window, drug resistance, and low cellular penetration. CNTs provide a unique opportunity to improve the drug delivery to the tumor, enhancing

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local accumulation in the desired site and reducing the toxic e ect of chemotherapy [165].

Relevant achievements in the field of nanotechnology to improve cancer treatment include carbon nanotubes loaded with classical therapeutic drugs and other anticancer agents like siRNA, chemosensitizers, radiosensitizers, and antiangiogenic compounds [166]. Liu et al. [167] developed hyaluronic acid (HA)-modi- fied amino SWVCNTs to target breast cancer cells overexpressing CD44, improving doxorubicin (DOX) deliver. In vitro studies in MDA-MB-231 breast cancer cells showed an increased intracellular DOX delivery, higher inhibition of proliferation and induction of apoptosis, and decreased cell migration.

The tubular structure of nanotubes is also optimal to attach DNA or RNA molecules to modulate the expression of specific genes [168]. In this sense, Taghavi et al. [169] functionalized SWCNTs with AS1411 aptamer as ligand to target tumor cells and with DOX and small interfering RNA (siRNA) molecules to perform chemotherapy and gene therapy, respectively. This nanoplatform increased cell death in a model of human gastric cancer measured in vitro when compared to the individual CNT-based treatments or with free DOX. Chang Guo et al. [170] prepared cationic MWCNT-NH3+ to deliver siRNA against polo-like kinase 1 (PLK1) in a lung carcinoma model in vivo. CNTs were injected directly in the tumoral mass, finding higher e ciency of PLK1 silencing compared to liposomes due to a higher CNT cell penetration.

The possibilities that carbon nanotubes o ers as drug carriers in the treatment of malignant tumors is very large [171–173], but they a ord other potential approaches in cancer treatment, like photothermal therapy (PTT) [174]. CNTs can be used as external agents in PTT because they absorb NIR radiation and e ciently convert it into heat energy, allowing the ablation of the cells in the CNT surroundings. Virani et al. used this phenomenon to target bladder cancer cells [175]. They conjugated SWCNTs with annexin V, which specifically binds to bladder cancer cells and tested the method in vivo. Once the CNTs reached the cancer, the tumor was heated using NIR light, inducing cell death and preserving the healthy bladder wall. They founded no damage on the bladder 24 h after treatment and no tumors were visible. To date, the main approach for PTT with CNTs has been based on the irradiation by laser or infrared light, limiting the application to superficial tissues due to low penetration capacity, but CNTs can also be exposed to external electromagnetic fields in a non-contact manner, achieving higher penetration while obtaining the desired thermal e ects [176].

Nowadays, chemoresistant tumors with a high tumor load and in advanced stages require the combination of di erent therapies for e ective treatment. CNTs are well suited for this, with carbon-based nanoparticles being used for combining thermal therapy with other therapeutic approaches based on drug delivery [177]. Wang et al. reported the use of NIR photothermal therapy and RNAi to enhanced tumor cells death in a prostate cancer model [178]. They synthesized SWCNTs functionalized with polyethylenimine to allow siRNA complexation, and decorated the CNT with a peptide motif that specifically binds to tumor cells. The cancer therapy e ect of these CNTs was tested in vitro and in vivo using PC-3 tumor cells. The CNTs silenced the target gen, causing significant inhibition

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of the tumor growth, and, when combined with photothermal therapy, the results showed a more e ective treatment.

4.2.2 Neurodegeneration Treatment

One of the big problems in the treatment of neurodegenerative diseases like Parkinson, dementia, or Alzheimer’s, is the presence of the blood–brain barrier (BBB). The BBB restricts the passage of bacteria and toxic molecules to avoid brain damage but also prevents the brain uptake of most pharmaceuticals, precluding the pass of drugs from blood circulation to brain tissue. In fact, neurological disorders belong to the group of pathologies with the greatest need of new technologies to improve diagnosis and provide an adequate therapy, and nanobiotechnology can help in both.

Al-Jamal’s laboratory proved [179–181] that CNTs are able to cross the blood–brain barrier without damaging it. They used in vitro cultures of primary porcine brain endothelial cells, which simulate the BBB, showing that MWCNTs functionalized with NH­3 crossed 13% of the layer in 72 h. They also demonstrated in vivo that 5 min after intravenous injection of MWCNTs-NH3 in mice, 1% of the injected dose crossed the BBB. In further experiments, they studied the impact of the diameter of the nanotube and the functionalization with angiopep-2 (AGP), a ligand for the low-density lipoprotein receptor related protein-1, in the extent of passing the BBB. They found that wider nanotubes linked with AGP showed a higher e ect on crossing the BBB than those not functionalized. Thinner CNTs did not depict any improvement in the brain uptake between functionalized and unfunctionalized MWCNTs, although they exhibited more uptake than the wider ones. Finally, they tested them in vivo using a GL261 glioma model, showing higher uptake for CNT functionalized with AGP. Costa et al. [182] took advantage of this property to develop an Alzheimer’s biomarker based on gadolinium. MWCNTs were functionalized with the Pittsburgh Compound B, a molecule that binds to the amyloid beta plaques present in Alzheimer’s disease and is currently used for imaging Alzheimer’s with PET. The group studied the in vivo distribution of the nanotubes, finding that they were able to cross the BBB, making them a good contrast agent to detect the presence of the plaques with MRI. Lohan et al. [183] reported the development of MWCNTs functionalized with berberine (BRB), an isoquinoline alkaloid used in dementia and other mental disorders, to evaluate the anti-Alzheimer’s potential of the drug. The authors injected the nanotubes in healthy male Wistar rats after intracerebroventricular administration of amyloid beta peptide. They proved that BRBMWCNTS crossed the BBB, reaching the plaques, and found a decrease of malondialdehyde and reduced glutathione, two of Alzheimer’s biomarkers.

The structural damage occurring in some neuro-diseases can also be minimized with carbon nanotubes. Hassanzadeh et al. [184] prepared MWCNTs-NGF complexed with nerve growth factor (NGF), a protein related to the survival and maintenance of neurons population, and investigated its e ect in an in vitro model of ischemia. The results were compared with those obtained with free NGF, finding higher protection for the new CNT-NGF complex.

As in cancer, CNTs found further applications in neurodegenerative diseases beyond drug delivery. CNTs can be used to activate brain cells due to their electrical

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