*2.2. Nanocarriers-IN-Nanofibers*

Nanocarriers have gained attention in drug delivery due to their ability to act as vehicles in the transport and delivery of different drugs. It is important to remark that nanocarriers are not only interesting in terms of vehiculization, they are also relevant because after introducing the drug into the nanocarrier, the amount of chemotherapeutic molecule to be delivered is considerably increased. It is accepted that a colloidal system is formed by a complex fluid where a certain substance (disperse phase) remains immersed in another substance (solvent). Accordingly, a colloidal system is formed by a disperse phase containing solid particles that are dispersed in a liquid. In this context, vesicles, micelles, microgels, or emulsions are colloidal structures where the disperse phase is a solid particle which is dispersed into a certain solvent. The possibility to introduce chemotherapeutic drugs into these colloidal particles has been extensively exploited in drug delivery applications and cancer treatments. Importantly, as these particles are in the range of nanometers, they can be incorporated into the human body by intravenous administration. However, it is important to mention that the simple introduction of drugs into some nanocarriers normally leads to inevitable burst drug release. To overcome this limitation, some improvements have been found, for example, by the incorporation of these nanocarriers into polymeric NFs, which improves drug delivery and the applicability of cancer treatments.

#### 2.2.1. Vesicles and Micelles as Nanocarriers

Vesicles and micelles have been used as drug nanocarriers to be incorporated into polymeric NFs. An important advantage of this type of colloidal particles is the fact that they possess in their structure two different environments (hydrophobic or hydrophilic) that can be exploited in, for example, the incorporation of two different drugs, thus performing dual drug delivery. Li et al. [33] investigated a dual drug delivery approach by using two types of drugs, 5-FU and paenolum. 5-Fluorouracil is a hydrophilic chemotherapeutic drug principally used in colon cancer treatment, and paenolum is a hydrophobic molecule used to prevent blood platelet clotting, thus having anti-inflammatory properties. Initially, the vesicles were fabricated by mixing two surfactants, cetyltrimethylammonium bromide (CTAB) and sodium dodecylbenenesulfonate (SDBS), which were able to trap the aforementioned drugs. As is represented in Figure 2A, hydrophilic 5-FU were encapsulated within the aqueous inner part of the vesicle, and the hydrophobic paenolum was situated into the external bilayer of the vesicle. Then, after nanocarriers fabrication and drug encapsulation, they were mixed with a PEO solution, and blend electrospinning was performed to obtain a core/shell scaffold, with the drug-loaded vesicles as core introduced into PEO NFs, Figure 2B. The release investigations concluded that the hydrophilic drug was released in an increased manner when the molar ratio of CTAB/SDBS was higher. In contrast, the hydrophobic drug showed a decrease in the release capability as the molar ratio of surfactants was increased. Figure 2A,B shows a schematic representation for the incorporation of dual drug-loaded vesicles into PEO NFs. The vesicle is initially introduced into the mixture of drugs, and then electrospinning is used to form the vesicle-IN-PEO NFs scaffolds. More recently, the same authors fabricated three different vesicles that were used as nanocapsules systems introduced into polymeric NFs for drug delivery purposes. These vesicles

were composed of didodecyldimethylammonium bromide, cetyl trimethyl ammonium bromide (CTAB)/sodium dodecyl benzene sulfonate (SDBS) (7/3) and CTAB/SDBS (3/7). After that, these vesicles were introduced into nanocapsules fabricated by a mixture of sodium alginate and chitosan. PEO NFs were obtained by blend electrospinning, and they were composed by PEO, containing a mixture of chitosan/sodium alginate with the vesicles as a template. In this work 5-FU was chosen as a model chemotherapeutic molecule to be incorporated during vesicles fabrication. The drug release behavior was followed by UV-visible spectroscopy. As was expected, the different drug-delivery systems showed different release rates and pH-responsive behaviors.

**Figure 2.** Schematic representation of the preparation of hydrophilic/hydrophobic electrospun composite fibers. (**A**) Accumulation of the drug mixture into the vesicle and (**B**) incorporation into the NF by electrospinning and drug release. Reprinted with permission from reference [33]. Copyright American Chemical Society, 2015.

Other types of linear polymer, as pluronic F127, were also used for release investigations, along with with tissue regeneration. Electrospinning was chosen for the fabrication of a scaffold containing high molecular weight PCL NFs containing pluronic (F127) vesicles, which were delivered by exploiting the slow dissolution of PCL into glacial acetic acid. The vesicles were fabricated by pluronic F127 self-assembled with low-molecular weight PCL in a tetrahydrofuran-water mixture [34]. The authors were able to tune the vesicle size from 1 to 10 μm in diameter. Time-dependent stability of the vesicles in glacial acetic acid was determined before the electrospinning process. The electrospun membrane was found to be composed of pluronic F127/PCL vesicles within a PCL mat with a fiber diameter between 50–300 nm. Authors proposed that the most probable condition for the vesicles generation is the non-solubility driven self-assembly and stabilization of PCL and F127 into bilayers in the tetrahydrofuran (THF)-water mixture. By using this method, the amphiphilic polymer is dissolved in a water miscible organic solvent and mixed at a high speed with water, leading to the rapid precipitation of polymers into nanoscale particles. Drug delivery of a model molecule as rhodamine-B (introduced into the polymer network) for this composite pluronic F127-IN-PCL showed an important reduction in the release rate of this molecule, when it was compared to the free vesicles. Indeed, the systems containing vesicles in the membrane presented an enhanced hydrophilicity compared to the control PCL membrane. Apart from drug delivery behavior, this increased surface hydrophilicity was exploited for increasing the cell viability of L929 cells on the membrane [34]. Another study with PCL was carried out by Yohe et al. [35] using the N-38 anticancer drug for the colorectal cancer cell line HT-29. They electrospun a mesh using 10% of a hydrophobic poly(glycerolmonostearate-co-ε-caprolactone) (PGC-C18) and 90% of PCL loaded with the N-38, showing promising results in the cytotoxicity of the cell line.

Micelles introduced into polymeric NFs have been also used for dual drug delivery. Hu et al. [36] fabricated colloidal structures formed by a block co-polymer composed by methoxypoly(ethylene glycol)-block-poly(L-lactide). Into these colloidal particles, a water soluble chemotherapeutic drug (5-FU) and a lipophilic drug as cefradine (an antibiotic active against Gram positive bacteria) were introduced. The external shell of the NFs was fabricated by a mixture of chitosan and PEO, and the micelles loading drugs were introduced into NFs by blend electrospinning. The NFs without micelles were uniform and smooth, with diameters in the range of 100–500 nm. However, after drug-loaded micelles incorporation within the NFs, the surface of the NFs was relatively rough, with diameters in the range of 200–800 nm, with some black spheres with diameters of approximately 150 nm in the NFs. By using this drug loaded nanocarrier-IN-NFs, they performed release investigations that revealed a low burst release tendency of 5-FU and Cefradin. Indeed, this system was able to reduce the activity of HepG-2 cells with good cell viability after 3 days of incubation. Zhang et al. [37] fabricated micelles composed by triblock copolymers as poly(L-glutamic acid)-b-poly(propyleneoxide)-b-poly(L-glutamic acid) which were able to deliver low water-soluble drugs, such as PTX, at clinically relevant doses. These micelles containing PTX were used as drug loaded nanocarrier which were grafted onto NFs scaffolds of (poly(L-lactide-co-ε-caprolactone) (PLCL):fibrinogen; 2:1 (w/w)) by blend electrospinning. Yang et al. [38] fabricated an implantable device structured as drug-loaded micelles-IN-NFs for controlled drug delivery. In this case authors used DOX introduced into micelles composed by PCL-PEG copolymer. The implantable devices were fabricated by coaxial electrospinning, with the core composed by the DOX-loaded PCL-PEG micelles introduced into a PVA solution dissolved in distillated water. The outer shell layer of the NFs was composed by genipin cross-linked gelatin. Importantly, these authors also functionalized the micelles with folic acid to specifically target tumor cells. The drug release investigations demonstrated that the implantable device reduced the drug dose, the frequency of administration and side effect of chemotherapeutic drugs while maintaining highly therapeutic efficacy against solid tumors.
