2.2.2. Silica Particles as Nanocarriers

Dual drug delivery has been also performed by using two different types of colloidal nanoparticles as mesoporous silica nanoparticles (MSNs) and hydroxyapatite nanoparticles. These colloidal systems were used as nanocarriers for the incorporation of DOX HCl and the topoisomerase inhibitor hydroxycamptothecin [39]. Initially, the mixture of mesoporous silica particles and hydroxyapatite nanoparticles individually containing both chemotherapeutic drugs was a mixture within a polymer solution of PLGA to be introduced into NFs as a core/shell structure by electrospinning. This dual anticancer biocompatible system improved the mechanical capacity as well as the thermal stability of the NFs. In addition, when in vitro investigation was performed, the dual micelle-IN-NFs system provided a sustained and controlled drug release and an improved capacity for inhibiting HeLa cells growth. Using a similar approach, Qiu et al. [40] fabricated a drug-loaded implantable device for the treatment of a tissue defect after tumor resection. They used MSNs as nanocarriers for the incorporation of the anticancer drug DOX hydrochloride. These MSNs colloidal systems with incorporated chemotherapeutic drugs (DOX@MSNs) were introduced into PLLA NFs generated by electrospinning, thus obtaining a drug-loaded NFs scaffold DOX@MSNs-IN-NFs. Initially they confirmed the successful introduction of DOX-loaded MSNs into the PLLA NFs by UV-vis spectroscopy, and then several nanocomposite systems with different MSNs and DOX contents were fabricated. Figure 3 shows the schematic representation for the fabrication of DOX-loaded MSNs-IN-PLLA NFs. Optimal results concerning particles distribution that also improved thermal stability were found by

using PLLA/1.0% DOX and 10% MSNs NFs. These authors investigated the in vitro antitumor efficacy against HeLa cells, and they found high DOX-loading capacities. Due to this fact, the drug was released in a sustained and prolonged manner, with a higher in vitro antitumor efficacy compared with free MSNs particles. Thus, these fabricated composite NFs mats are highly promising as a local implantable device for potential postsurgical cancer treatment. Yuan et al. [41] propose a drug delivery system which can release anti-tumor drugs in two phases. They designed a NFs scaffold for breast-conserving therapy after breast cancer by using DOX-loaded MSNs into an electrospun PLLA nanofibrous scaffold. In vivo results (mice) showed a significantly inhibition in the tumor growth, making its use promising in a coadjuvant therapy against this tumor type.

**Figure 3.** Schematic illustration for the process of fabrication of DOX@MSNs-IN-NFs electrospun composite NFs and the location of DOX in the fiber [40].

#### 2.2.3. Gelatin Nanoparticles as Nanocarriers

Song et al. [42] used gelatin nanospheres (GNs) as colloidal systems to improve the antibacterial effects of silk NFs membranes. They prepared two types of GNs by adding different amounts of glutaraldehyde into the suspension during the synthesis of GNs. The size and distribution of the GNs into the NFs, fabricated by electrospinning and using a mixture of PEO and silk fibroin, was monitored by fluorescent labeling. Positively charged drugs as vancomycin and colistin were used as a model for controlled drug delivery investigations. Vancomycin is an antibacterial compound that inhibits the synthesis of the bacterial cell wall, and colistin is an apolypeptide that is effective against most of the Gram-negative bacilli. These authors used both blend and coaxial electrospinning to fabricate GNs-IN-PEO/silk NFs, and they examined the antibacterial effect by introducing the mentioned antibacterial drugs. By using the NGs synthesized with the highest amount of glutaraldehyde, the fabricated NFs supplied a more sustained release of vancomycin compared with pure GNs. Indeed, apart from being totally cytocompatible, the NFs showed excellent and sustained antibacterial effects against Staphylococcus aureus. Lai et al. [43] fabricated collagen (Col) and hyaluronic acid (HA) NFs with the aim to release a series of growth factors directly embedded in the NFs or encapsulated in the gelatin nanoparticles (GNs) by using electrospinning technology. The fabricated GNs-IN-Col/HA NFs showed mechanical properties that mimicked human natural skin. The designed GNs-IN-Col/HA NFs were able to release growth factors in a slow controlled manner for up to 1 month. From the above, the electrospun Col-HA-GN composite nanofibrous skin substitute with a stage-wise release pattern of multiple angiogenic factors could be a promising bioengineered construct for chronic wound healing in skin tissue regeneration. They also used several release patterns for GNs-IN-Col/HA NFs with 4 different growth factors and demonstrated their potential capability to deliver multiple bioactive molecules.

Electrospun PLGA and PLGA/gelatin NFs embedded with MSNs were synthesized, obtaining MSNs distributed in the core of the fiber. PLGA and MSNs contributed to increase the hydrophobicity of electrospun NFs and the gelatin contributed to increase the mechanical properties of this scaffold. With a final size of approximately 267 nm, these nanoformulations are synthesized to provide a very suitable microenvironment for the adhesion, growth and migration of stem cells involved in nervous tissue regeneration [44,45]. Aytac et al. [46] synthesized electrospun gelatin NFs to vehiculate ciprofloxacin (CIP) and hydroxypropyl-beta-cyclodextrin (HPβCD)-inclusion complex (IC), which is normally used to improve the physico-chemical properties of some drugs as well as their bioavailability. This IC achieved an increase of the solubility and wettability of the nanoformulations, leading to their fast dissolution and therefore to a fast release of CIP transported in gelatin NFs, which can be an important property in certain situations where a rapid release of drugs is required. With this type of gelatin-based scaffolds, in addition to drug transport and use for tissue engineering, the migration of certain cells can also be induced or favored. This is the case in the study of Piran et al. [47] that synthesized electrospun three-layered scaffold with plasma enriched with growth factor to promote the migration and growth of fibroblast, which is a very important aspect in the regeneration of wounds.

### 2.2.4. Stimuli-Responsive Nanoparticles as Nanocarriers

Some stimuli-responsive colloidal particles have been used as nanocarriers to be introduced into polymeric NFs for drug delivery applications. For example, Gong el al. [48] introduced redox-responsive nanoparticles into a polymeric scaffold to be incorporated into the body with the aim to deliver a growth factor (morphogenic protein BMP-2) used of bone regeneration. The strategy to fabricate redox-sensitive NFs with a core/shell structure consisted of a blend of PCL and redox responsive c-6A PEG-PCL nanogel with –S–S– bond on the outer shell. This redox-sensitive shell was able to respond to the change of the glutathione concentration and thus regulate the BMP-2 release for in vitro and in vivo investigations.

Light-responsive nanoparticles, as TiO2 and incorporated into a polyacrylonitrile (PAN)/multiwalled carbon nanotube composite NFs have been reported for the photocatalytic degradation of pharmaceutical molecules as Ibuprofen, Cetirizine, and Naproxen [49]. Visible light (0.1 W/cm2) irradiation was employed to investigate the drug degradation. The photocatalytic degradation of molecules using TiO2-IN-PAC/MWCNT NFs was higher compared with TiO2-IN-PAC NFs under visible light irradiation. A total degradation of drugs molecules was performed at 200, 50, and 90 min, respectively under visible light.

Another interesting strategy was developed by Elashnikov et al. [50]. They were able to release antimicrobial molecules from thermos-responsive microgels introduced into PLLA NFs. Crystal violet (CV) was incorporated within the polymer network of temperature-responsive PNIPAM microgels used as nanocarriers. Then, blend electrospinning was performed using PLLA as a biocompatible polymer, thus resulting in composite PLLA NFs with incorporated pNIPAM particles containing antimicrobial CV. They investigated the controlled drug delivery behavior of these core/shell NFs by UV-vis spectroscopy, which was produced after modification of the external temperature below and above the lower critical solution temperature (LCST). The antibacterial activity was investigated against gram-negative Escherichia coli (E. coli) and gram-positive Staphylococcus epidermidis (S. epidermidis). Authors demonstrated that the temperature-responsive release of antibacterial CV possessed remarkable antibacterial activity. This activity showed higher inhibition zones at temperatures above the LCST, with its size dependent on the polymers ratio and temperature.

### *2.3. Stimuli-Responsive Nanofibers*

As was mentioned, synthetic polymers have been extensively used as scaffolds in the synthesis of NFs for drug delivery investigations. However, during the last years, important researching efforts have been focused on the use of stimuli-responsive systems [20]. These structures can undergo changes in response to external stimulus, as temperature, pH, ion strength, or solvent nature [51–53]. Most of the recent studies in the area of switchable drug release have been dedicated to the creation of systems for drug encapsulation based on two types of polymer [54]: pH-responsive and thermo-responsive polymers.

### 2.3.1. pH-Responsive Nanofibers

The acidic environment found in tumor tissues can be employed as a way to specifically target the release of antitumor drugs at the tumor in response to a change in pH by the use of nanoformulations sensitive to pH changes [55]. Illangakoon et al. [56] used ES100, which is an anionic co-polymer constituted by metacrylic acid and methylmethacrylate, for the fabrication of pH-responsive nanofiber for the delivery investigation of 5-FU. Co-axial electrospinning was carried out with a core composed of poly(vinylpirrolidone), ethyl cellulose (EC) and the 5-FU drug, and the shell was formed from pH-responsive ES100. The drug release investigation demonstrated a controlled drug release developed at pH 1, reaching a maximum of 80% drug release after 2 h, produced by the diffusion of 5-FU through the pores of the ES100 polymer. At this pH, the polymeric fibers were fragmented, supplying an increased 5-FU delivery. Indeed, these authors fabricated NFs with a core/shell structure made of ES100 for the shell and Eudragit L100 (EL100) for the core to allow the controlled release at certain pH conditions, and controlled this release using the cover thickness [57]. Tran et al. [58] introduced ibuprofen into pH- and thermo-responsive polymers for controlled drug delivery investigations. Ibuprofen was initially mixed with a polymeric solution of PCL as the control experiment and also into poly(Nisopropylacrylamide-co-methacrylic acid) (pNIPAM-co-MAA) to fabricate stimuli-responsive NFs by blend electrospinning. As comparative results, when PCL NFs were investigated as a drug delivery system for ibuprofen, the fabricated NFs did not show significant drug release behavior at temperatures between 22–40 ◦C or pH from 1.7–7.4. However, NFs generated from pNIPAM-co-MAA were able to diffuse ibuprofen in a linear and controllable manner when the temperature was above the lower critical solution temperature (LCST) of pNIPAM-co-MAA (33 ◦C), as well as at pH lower that the pKa of carboxylic acids (pH 2). However, when the drug delivery experiments were performed at room temperature, the release rate was radically increased by closely ten times, compared to the release behavior at higher temperature and lower pH. NFs of cationic chitosan and poly(acrylic acid) (PAA) were synthetized with different levels of Cs deacetylation, showing that the mechanical properties of these NFs are determined by both the pH and the level of deacetylation, which could be useful in biomedical applications such as the transport and release of drugs [59]. Gelatin and poly(lactide-co-ε-caprolactone) (PLCL) were used to synthetize NFs that were loaded with ciprofloxacin and sodium bicarbonate with a response to low pH of gelatin/sodium bicarbonate fibers, whereas the hydrophobic PLCL had no sensitivity to pH [60]. These NFs not only showed good biocompatibility in fibroblasts (L929), they were also able to stimulate cell growth compared to untreated cells. Functionalized electrospun PCL scaffolds sensitive to pH changes, were loaded with DOX and tested at different pH levels (from 7.4 to 2.5) finding the highest drug release (90%–95%) at the lowest pH levels [61]. This was observed in the human embryonic kidney cells (HEK) treated with these scaffolds with a cell viability at pH 6 lower than those obtained at pH 7.2.

Thixotropic silk NFs hydrogels were loaded with DOX and it was designed to release the drug in the tumor site due to their thixotropy capability [62]. This allows the hydrogel inoculation that then solidifies at the specific site and releases the drug in response to certain pH conditions. In the in vitro studies carried out on the human breast cancer cell line MDA-MB-231, it could be observed that the DOX-loaded hydrogels were more suitable for a long treatment, since even after 10 days they continued to inhibit cell growth, in contrast with free DOX. In the in vivo studies in breast tumors bearing BALB/c nude mice, it was observed that after the inoculation of the liquid hydrogel it solidified around the tumor, finding remains of the hydrogel even 5 weeks after the inoculation. In a similar way, in the in vivo studies after the inoculation, the decrease in tumor volume was similar with the treatment of free DOX and DOX transported by the hydrogels in the first weeks. However, at the

fifth week, significant differences were observed in the volume and weight of the tumor treated with DOX-loaded hydrogels compared to free DOX, reaching reductions of approximately 1.5 times for both parameters, see Figure 4.

**Figure 4.** Stimuli-responsive NFs. Once NFs are synthesized by the electrospinning process and loaded with the antitumor drug, treatment may be applied in an experimental mouse model that carries a specific type of tumor. Once the treatment has been inoculated, an internal stimulus, such as the low pH present in the tumor tissues, or an external stimulus such as a temperature rise, stimulate the release of the drug at the specific site of the tumor, thus applying the treatment on tumor cells. Reprinted with permission from reference [62]. Copyright American Chemical Society, 2016.
