2.4.1. CNTs/Nanofibers

Carbon nanotubes have been used as platforms to improve mechanical, structural and drug delivery properties in NFs synthesized by electropun. In a recent investigation carried out by Qi et al. [73], DOX was chemically incorporated onto the surface of MWCNTs. After being optimized, the drug encapsulated up to 83.7% and the dispersion of MWCNTS@DOX particles was mixed with a PLGA polymer solution at 3 different amounts of DOX, relative to PLGA, to fabricate a composite NF by electrospinning. Importantly, the incorporation of MWCNTs into PLGA NFs did not alter the structure of the PLGA NFs, instead it improved their mechanical properties. In vitro viability assay demonstrated that the developed DOX-loaded MWCNTs-IN-PLGA composite NFs were totally cytocompatible with L929 cells. The drug delivery investigation confirmed that this composite system was able to reduce burst DOX release, and it also allowed a continued DOX release over 42 days. This composite structure structured as CNTs-IN-NFs was also used for Yu el al. for the incorporation of DOX HCl thus creating a hybrid NFs mat by blend electrospinning. In this case CNTs were also used as carriers of DOX and they were introduced within PLGA NFs by electrospinning, thus creating a composite nanofibrous mat. In this investigation, authors modified the amount of CNTs in the final mixture polymer-CNTs during electrospinning. The in vitro antitumor efficacy against HeLa cells was investigated, resulting in a DOX release by a sustained and prolonged manner, which effectively inhibited growth of HeLa cells. Figure 5 shows a schematic representation for the fabrication of DOX-loaded CNTs-IN-PLGA NFs by blend electrospinning.

**Figure 5.** Schematic illustration for the fabrication process of PLGA/DOX-IN-CNTs electrospun composite NFs. The morphology and diameter distributions of PLGA and PLGA/DOX@CNTs composite NFs [74].

Zhang et al. [75] fabricated a new class of highly porous NFs by using PLA as the principal polymer scaffold and PEO as a porogen. Different concentrations of CNTs were incorporated onto the fibers to allow self-sealing behavior, which was carried out by photothermal conversion after light irradiation. Basically, they developed a strategy to fabricate porous PLA/CNT fibers with controlled pore sizes. Indeed, CNTs were used in the current study to perform the pore self-closure through their photothermal conversion ability. Zhang et al. investigated several ratio polymer/CNTs during the electrospinning process and they found that the fibers containing 0.4 mg/mL of CNTs showed the optimum encapsulation efficiency of model biomacromolecules such as dextran, bovine serum albumin, and nucleic acid. The pores of the surface were reversibly reopened by PLA degradation, reaching

a stable release of biomacromolecules after encapsulation. They showed morphological changes of PLA/CNT fibers with 0, 0.2, 0.4, and 0.8 mg/mL CNT concentrations. At a CNT concentration of 0.2 mg/mL, only a small amount of CNTs could be seen on the fibers surface. As the concentration was increased to 0.4 mg/mL, much more CNTs were observed, which seemed to be dispersed homogeneously around the nanopores on the fibers. Nevertheless, with further increase of the CNT concentration to 0.8 mg/mL, significant agglomeration was observed. After trapping the drug molecules, a controlled and sustained release from the fibers over extended periods of time was investigated. An extended release of the molecules for over the period of 15 days was achieved, with a relatively low initial burst release within the first 24 h. Indeed, in these structures the amount of CNT introduced into the nanofiber was relevant to control parameters as morphology, structure, thermal/mechanical capability, degradation, and cell viability.

It is well-known that CNTs have the capacity to acts as thermal generators by absorbing near-infrared radiation (NIR). Zhang et al. [76] combined MWCNTs and DOX in a methanol/chloroform solution with PLLA dispersed in CHCl3 to fabricate NFs by the electrospinning technique. Without NIR irradiation, DOX release was extremely restricted. However, when NIR irradiation (2 W/cm2) was applied on DOX-loaded MWCNTs-IN-PLLA NFs burst release of the loaded DOX was observed at the time of 2 h with an amount of ~20% during the 30 min irradiation period. Indeed, these authors demonstrated that after NIR radiation, the temperature of the tumor area in contact with the NFs was significantly increased. Apart from that, these multifunctional NFs showed increased cytotoxicity both in vitro and in vivo for Hela cancer cells through the combination of photothermal induced hyperthermia and drug delivery. Barzegar et al. [77] introduced graphene into PVA NFs by electrospinning, thus creating grapheme/PVA hybrid NFs. Figure 6 shows scanning electron microscope (SEM) images where uniform hollow PVA NFs containing graphene dispersion within the NFs can be appreciated. The synthesized polymer reinforced NFs have potential biomedical materials for drug delivery.

**Figure 6.** SEM micrographs of PVA/(graphene foam and expanded graphite) NFs at 2 kV operating voltage for (**A**) solution with 0.02 g GF concentration, (**B**) solution with 0.08 g GF concentration, (**C**) solution with 0.02 g EG concentration and (**D**) solution with 0.08 g EG. Reprinted with permission from reference [77]. Copyright Elsevier, 2015.

In recent years, there has been an increase in the number of investigations related to the use of electrospun NFs combined with conductive nanomaterials, as biosensors, due to their promising applications [78]. Electrospun NFs in combination with CNTs have also been investigated as biosensors for the detection of early stages of pancreatic cancer by the use of the biomarker CA19-9. Electrospun NFs made of poly(allylamine hydrochloride) and polyamide 6 were covered with MWCNTs, and CA19-9 detection were determined by electrochemical impedance spectroscopy [79]. This biosensor showed a good sensitivity for the antigen without the rest of the blood components could suppose interference in the detection. This high sensitivity was achieved through antibody-antigen irreversible adsorption. In addition, this system was able to distinguish between patients with higher levels of the biomarker in blood and those who had it in lower concentration.

However, concerning toxicology studies, it has been reported that CNTs produce some adverse effects, due principally to the incorporation of catalytic metal impurities during their fabrication [80]. For this reason, a general use of CNTs in the biomedical field has generated some doubts about their security and possible toxicity. For example, long-term accumulations of CNTs were produced in lungs and liver of mice after exposure during 90 days [81]. Some reported investigations detailed the negative effects of these CNTs in biological systems. An alternative to reduce toxicity is the use of surface functionalized CNTs. Specifically, a recent investigation showed that pure CNTs had negative effect on the systemic immunity, producing more inflammation and immunosuppression as compared to some surface modified CNTs, such as PEG-modified CNTs [82].

#### 2.4.2. Magnetic Nanoparticles/Nanofibers

Magnetic nanoparticles are another type of colloidal particles widely used in biomedical applications. The systems, in the range of nanometers, are able to respond to external magnetic fields. This property has been extensively exploited in the biomedical field for localized drug delivery, because they can be transported to a specific area by the application of an external magnetic field. Apart from conventional drug delivery systems used in nanomedicine for the reduction of solid tumors, magnetic hyperthermia treatments [83–85], in which magnetic or magnetic-derived NPs are administered to tumors, and then a local heat is supplied under alternating magnetic field (AMF) application, have attracted an ever increasing interest due to their improved precision for cancer therapy [86–88]. The basis of using hyperthermia as a treatment modality for cancer is the high sensitivity of cancer cells to temperatures in the range from 41 to 45 ◦C, in contrast to normal cells. However, the use of free magnetic nanoparticles has certain limitations such as poor tumor targetability, high variability in the amount of magnetic nanoparticles administered to the tumor, as well as the transfer of magnetic nanoparticles into the healthy tissues close to the tumor. To overcome this disadvantage, magnetic NPs have been also used in electrospinning to fabricate hybrid NFs for drug delivery and cancer treatment purposes.

It is important to mention that a significant advantage of magnetic nanoparticles is that they present a reduced toxicity, while also being accepted by the human body. Indeed, once they are placed into the cells they degraded reasonably quickly [89]. The degradation of magnetic nanoparticles into iron and oxygen is performed inside the lysosomes of macrophages, and it is influenced by several parameters as the presence of hydrolytic enzymes, a low pH, as well as proteins related with the iron metabolism. In particular, iron oxides nanoparticles are able to degrade in vivo by iron mobilization and some other published routes [90]. Most importantly, magnetite is an iron oxides derivative approved by FDA for in vivo investigation [91].

Feng et al. [92] prepared a mixture of Fe3O4 NP and graphene oxide sheets containing functional groups to be introduced into PAN NFs for guiding cellular application. They used blend electrospinning to fabricate a hybrid GO/Fe3O4-IN-PAN as short-fibers (SFs). These synthesized NFs films were cut into small pieces, and after being dispersed in tert-butanol solution, they demonstrated a strong magnetic capability. As guiding cellular behavior, breast cancer cells were cultured on the surface of these magnetic SFs, and due to the external GO on the surface of SFs they promoted adhesion of cell membrane proteins and good biocompatibility. Indeed, guided cellular behavior by magnetic actuation with the help of magnetic SFs was performed. Huang et al. [93] introduced 50 nm iron oxide nanoparticles into polystyrene (PS) NFs scaffolds. Fe3O4 NPs were initially dispersed and ultrasonicated in THF, this dispersion was a mixture with a PS solution to proceed with blend electrospinning. Upon applying an AMF on these hybrid Fe3O4-IN-PS NFs, an important heating process occurred due to the high

loading capacity of the fibers. Using this hybrid structure, the fabricated magnetic fibers can be heated several times without loss of heating capacity or releasing magnetic nanoparticles. Indeed, these authors functionalized the surface of the hybrid NFs structure with collagen in order to increase cell attachment. In vivo investigations were performed by using Human SKOV-3 ovarian cancer cells, which were incorporated onto the fibers. After the application of an AMF during 10 min to the mats, the cancer cells deposited on the Fe3O4-in-fibers were eliminated. This methodology possesses two important advantages to be implemented for in vivo investigations, as the fibers can be loaded with magnetic nanoparticles in a controlled manner and the composite scaffolds can be localized in the body by magnetic resonance imaging (MRI). Sasikala et al. [94] fabricated an implantable hybrid magnetic NFs device to be applied in both magnetic hyperthermia, upon an AMF, and cancer cell-specific drug release, to perform a synergistically cancer therapy. A borate-containing anticancer drug was investigated known as bortezomib (BTZ), a protease inhibitor frequently used in chemotherapeutic administration. This device was fabricated by blend electrospinning, by a mixture that initially was a Fe3O4 NPs dispersion and a PLGA solution. Then, a shell of polydopamine was grown through a simple immersion method to be used as a shell-mimicking mussel adhesive. The mussel-inspired magnetic NFs with numerous catechol moieties were able to bind and release borate-containing anti-cancer drugs. Figure 7 includes transmission electron microscopy (TEM) image of free Fe3O4 NPs as well as field emission scanning electron microscopy (FESEM) and TEM images of electrospun PLGA NFs. The FESEM images confirmed that hybrid NFs shows good fiber morphology and some aggregation of the Fe3O4 NPs inside of the NFs. They investigated the effect of repeated hyperthermia application in murine breast cancer (4T1) cell lines by using the BTZ-loaded Fe3O4-IN-PLGA NFs. After three hyperthermia cycles (15 min/24 h) were applied to the BTZ-loaded Fe3O4-IN-PLGA NFs, an improved antitumor efficacy was obtained.

**Figure 7.** (**A**) TEM image of the iron oxide NPs (the inset shows the corresponding selected-area electron diffraction pattern), (**B**,**C**) FESEM images of electrospun PLGA (poly lactic-co-glycolic acid) NFs and magnetic NF matrix, respectively, (**D**) TEM image of MNF. Reprinted with permission from reference [94]. Copyright Elsevier, 2016.

In other reported cases, polyurethane NFs combined with superparamagnetic iron nanoparticles (γ-Fe2O3) have been synthesized for cancer treatment by hyperthermia, which were able to reach a temperature increase of up to 43 ◦C in 70s by the application of an AMF. This promising result was achieved by making the electrospinning process much more precise with the addition of a conical aluminum auxiliary electrode [95]. Radmansouri et al. [96] designed DOX hydrochloride-loaded electrospun chitosan/cobalt ferrite/titanium oxide NFs to treat melanoma cells (B16F10) by means of chemotherapy and hyperthermia at the same time. NFs were made of chitosan by the electrospinning process and were combined with cobalt ferrite nanoparticles and titanium oxide nanoparticles (used to modulate the temperature increase). Higher cell death was observed when hyperthermia and chemotherapy were combined, achieving a synergistic effect, which enhances the cytotoxic effect and allows a reduction of side effects.
