2.3.2. Thermo-Responsive Nanofibers

In general, there are various protocols for synthesizing nanoformulations sensitive to temperature that are suitable for their possible use in biomedicine, because they can be administered by injection and may be degraded. These properties are useful in the transport of drugs for cancer targeting and controlled release such as degradable NFs fabricated by an electrospinning technique [63]. Slemming-Adamsen et al. [64] presented a novel approach to introduce DOX into thermoresponsive pNIPAM-NHS/gelatin NFs by cross-linking with 1-ethyl-3-(3-dimethyl-aminopropyl)-1-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). This strategy consisted of a mixture with a solution of pNIPAM-NHS/gelatin acting as a shell with another mixture of EDC and NHS in the presence of DOX. By using this approach, EDC initiates the conjugation by bonding with a carboxyl group of the polymer. Then, the EDC-polymer conjugate is able to react with a primary amine, or, NHS, replacing EDC with the amine ester linkage. Finally, NHS can be replaced by a primary amine, linking the carboxyl-polymer with the amine-polymer. This mixture was electrospun to obtain cross-linked pNIPAM/gelatin NFs containing an anticancer drug that can be released in a controlled manner. The DOX-IN-pNIPAM NFs showed thermo-responsive swelling/deswelling properties. Indeed, the fabricated cross-linked NFs were able to release DOX when the temperature was raised above the LCST and were able to reduce the viability of human cervical cancer cells [64].

Zhang et al. [65] fabricated a core/shell structure formed by polylactic acid PLA as a core using electrospinning, and then a thermoresponsive pNIPAM shell was incorporated by UV photo-polymerization. Initially, biodegradable PLA NFs were fabricated by electrospinning in the presence of Combretastatin A4 (CA4), a tubulin polymerization inhibitor which was used as the model drug was produced. These fabricated PLA NFs were introduced into a pNIPAM solution in presence of the crosslinker (*N*,*N* -methylenbisacrylamide). After exposed to UV radiation, the drug-loaded PLA core was coated and cross-linked with a pNIPAM shell. The composite NFs exhibited different wettability confirmed by water contact angle measurements at temperatures below or above the lower critical solution temperature (LCST) of pNIPAM. Most importantly, in vitro drug release investigations demonstrated a difference drug release when the temperature was at 25 or 40 ◦C. For example, the pNIPAM shell could limit the release rate of CA4 below the LCST, however, above the LCST, the rate of drug release increased significantly. Cicotte et al. [66] used thermos-responsive pNIPAM films fabricated by electrospinning to exploit a rapid reversible adhesion of mammalian cells, thus performing cell attachment and detachment using pNIPAM scaffolds. These authors modified various parameters during the electrospinning process such as the needle gauge, collection time, and molecular weight of the polymer. Two types of cells were investigated for reversible attachment of pNIPAM mats that provided potential results by seeding mammalian cells from standard cell lines (MC3T3-E1) as well as cancerous tumor (EMT6) cells. Once attached, the temperature of the cells and mats was changed to ~25 ◦C, resulting in the extremely rapid swelling of the pNIPAM NFs. The authors found that pNIPAM mats fabricated using small and dense fibers fabricated from high molecular weight pNIPAM polymers are extremely appropriate as a rapid release method for cell sheet harvesting. Recently, new nanoformulations that allow the release of drugs in a dual way in response to both temperature and pH stimuli have been designed. This is the case of poly(N-isopropylacrylamide-co-acrylic acid) NFs in a passive thermoplastic polyurethane (TPU) which are sensitive to pH and temperature. Consequently, by varying these two parameters, the movement in terms of direction and size can be modulated, which could be interesting in several biomedical applications, such as drug release [67]. In another study, a thermo-sensitive polymer, PNIPAAm and a pH-sensitive polymer, Eudragit® L100-55 (EL100-55), were synthetized and made NFs by electrospinning [68]. These NFs showed sensibility to pH and temperature and a release of ketaprofen that are dependent on these parameters without toxicity against fibroblast, even at high concentrations. Another example is a fiber mixture of poly(N-vinylcaprolactam) and ethyl cellulose (EC) in the case of temperature-dependent release and EL100 fibers for pH-dependent release, synthesized by twin-jet electrospinning [69]. This mixture of fibers showed a sustained release of the non-steroidal anti-inflammatory drug ketoprofen in response to pH and changes in temperature, also showing a very good biocompatibility in fibroblasts. The study of biocompatibility of these NFs is noteworthy because it is an essential property that nanomaterials which are intended for therapeutic use must comply with. For this purpose, fibroblasts (L929) were seeded on cover slips that were previously sterilized and where fibers were directly slectrospun. After 1, 3 and 5 days of exposure, cytotoxicity is determined by the MTT assay. NFs are inclined to show good biocompatibility, but some types may be more appropriate than others, such as thermosensitive fibers made of poly(di(ethylene glycol) methyl ether methacrylate) (PDEGMA) synthesized and electrospun into fibers using EC, which showed great biocompatibility even after 5 days of exposure and better in vitro biocompatibility than other nanoformulations such as EC/NIPAM [70,71]. Another type of stimuli-responsive NFs that could be used for the transport of antitumor drugs is electrospun self-immolative polymer (SIP)/polyacrylonitrile (PAN) fibers [72], which depolymerize surprisingly rapidly in response to an external stimulus, producing an instantaneous release of the transported molecules at the right time.

#### *2.4. Hybrid Nanofibers*
