5.1.9. Miscellaneous

Among various miscellaneous drugs, caffeine is the most used agent worldwide that binds to adenosine receptors by mimicking natural adenosine, affecting various body functions. At present, electrospinning techniques are being widely used to fabricate caffeine loaded nanofibers as a FDDST facilitating a high surface area and high porosity of nanofibers that can lead to fast-wetting features of the nanofiber surface to ensure quick release properties of incorporated drugs. In these respects, drugs are usually encapsulated into nanofibers either in amorphous or nanocrystal format, ensuring high solubility, dissolution, rapid onset of action and bioavailability of loaded drugs. A study was designed by electrospinning using PVA as the drug nanocarriers of caffeine (CA) and riboflavin (RFN) in which the dissolution time and wetting time were 1.5 s and 4.5 s respectively for both PVA-CA and PVA-RFN. In addition, the release percentages of CA and RFN from PVA filament-forming nanofibers were 100 and 40 respectively within 1 min [96]. In another study, paracetamol and caffeine-loaded PVP electrospun nanofiber scaffolds were fabricated as a FDDST model with more than 90% drug loading capacity in which both loaded drugs remained intact even after electrospinning. This study indicated a high disintegration rate of the drug-loaded nanofiber scaffolds within 0.5 s whereas it took less than 150 s to reach 100% dissolution rate. The drug-loaded nanofiber matrix revealed its potency over the pure standard drugs (caffeine and paracetamol individually) [101]. Moreover, it was evidenced that electrostatic spinning has the potential to fabricate such nanofibers that can ensure the ultrafast drug delivery and dissolution of water-insoluble miscellaneous drugs even through the oral route of administration [102].

Discovering an effective and potential ophthalmic drug delivery system (ODDS) is still a challenge due to the quick elimination of ocular drugs like eye drops. Thus, rapid peroneal losses of eye drops, even with the high volume of administration resulting in low bioavailability and very short half-life of applied drugs and a lower amount of ophthalmic drugs (1–3% of total volume), can reach into intraocular tissues via penetration through the cornea. To address these problems, Gagandeep et al. (2014), developed a nanopatch using PVA and PCL via electrostatic spinning to load and deliver a combination of timolol maleate and dorzolamide hydrochloride as a model drug against glaucoma. As-developed drug-loaded nanopatches were 200–400 d·nm with almost 100% drug entrapment efficiency—no interaction has been found between polymers and encapsulated drugs. Furthermore, the in vitro drug release experiment alluded an initial burst release of incorporated drug from nanopatches followed by controlled release behavior for up to 24 h [103]. The overall results reveal the efficiency of electrospun nanopatches in ocular drug delivery.

Drug enrichment on the surface of the polymer is only possible if the drug is blended with the polymer solution before electrostatic spinning. This system facilitates an initial burst release of the loaded-drug resulting in rapid onset of action, but it also decreases the potential lifetime of delivery carriers. Hence, to obtain desired results such as sustained therapy, local and controlled release, enhanced activity and retrenchment of side and adverse effects, core-shell nanofibers would be the best option. One study was designed to load brimonidine tartrate (BT) into a first dissolving dendrimer nanofibers (DNF) against glaucoma (Figure 5a). The in vivo intraocular pressure (IOP) was evaluated as an efficacy parameter for both BT and BT-DNF (Figure 5b). As shown in Figure 5c, the single dose responses of BT and BT-DNF were almost similar to each other regarding the therapeutic effects. Nevertheless, after daily treatment with BT and BT-DNF over a period of 3 weeks, BT-DNF was more effective in comparison with BT for reducing IOP (Figure 5d) [58]. In another study, a novel method was established, combining both coaxial electrospinning and emulsion electrospinning to fabricate a core/shell fiber matrix. As-proposed PLGA coaxial-emulsion electrospun fibers were loaded with an anticonvulsant class of drug—Levetiracetam—and the aim was to implant them in the brain to treat various seizures in adult patients and children with epilepsy. The constant and linear release kinetics of the incorporated lower molecular weight drug Levetiracetam from fiber scaffolds was noticed during 480 h of the study [104].

**Figure 5.** (**a**) An illustrative diagram of nanofiber-based ophthalmic drug delivery system; (**b**) Ocular deposition of dendrimer nanofibers (DNF). Brown Norway rats (BNR) (n = 3) received a DNF-FITC mat topically in the right eye (experimental eye), whereas the left eye received no treatment (contralateral eye); (**c**) In vivo single dose response. BNR (n = 4) received a single dose of brimonidine tartrate (BT) topically via saline eye drops or DNF; (**d**) In vivo 3-week daily dose response. Brown Norway rats (n = 4) received a daily dose of BT via saline eye drops or DNF for 3 weeks. (Here in, Figure 5b–d reproduced from ref. [58] with permission from ACS Publication, 2017).

#### *5.2. DNA and RNA Delivery*

The delivery of nucleic acid molecules such as DNA or interfering RNA, into a target cell to knockout/ knockdown mutated gene expressions, either by gene editing or impeding the mechanism of the mutated gene, has proven its novelty in the field of regenerative disease treatment. In this case, when a gene misbehaves in a protein, gene therapy is able to introduce the new copy of that specific gene for recuperating the function of that protein by adjusting the signal transduction pathway [105]. To date, the nucleic acid delivery system depends on the carriers and can be folded into two groups; a viral and a non-viral vector. As nucleic acid directly implanted into the cell does not work, researchers use vectors as their carrier. In viral vectors, useful viruses are genetically modified with a specific nucleic acid so that they can enter into the target cell and introduce new genetic material in the place of missing or faulty genes. However, viral vectors are very specific such as their delivery strategy depends on the type of tissues and gene, even they can carry a very small size of a gene that may sometime cause mutation. In comparison with a viral vector, non-viral vectors are more appreciable among scientists owing to their manageable toxicity, ability to carry various size of genes, large surface area, and large porosity [106]. Among all types of non-viral vectors, nanofiber scaffolds are widely used even though problems associated with electrospun nanofiber scaffolds, such as inappropriate nucleic acid encapsulation and transfection efficiency, are still unsolved. To overcome these limitations, several attempts have been introduced, for example, core/shell, surface modification, coating, encapsulation, incorporation, or interfacing electrostatic interaction, to protect the nucleic acid [107–132].

However, the encapsulation of a nucleic acid/polymer complex in the core of core/shell nanofiber scaffolds can protect the encapsulated DNA or interfering RNA from biological degradation and denaturation, as well as prolong their release up to several months by controlling it. For instance, non-knitted, membraneous nanofiber scaffolds were fabricated via the electrospinning procedure as a gene delivery carrier, containing plasmid DNA encapsulated with poly(lactide-co-glycolide) and poly(D,L-lactide)–poly(ethylene glycol) type biodegradable synthetic copolymers [107]. These promising nanofiber scaffolds can control the release behavior of plasmid DNA over 20 h of the study, whereas the burst release of plasmid DNA appeared within 2 h of the study. In addition, the cumulative release profile indicated up to 80% of plasmid DNA was released from nanofiber scaffolds. The released plasmid DNA had a high cellular transfection efficiency with specific protein encoding properties. In another study, various fiber mesh scaffolds were designed and prepared through the

coaxial electrospinning method containing the plasmid DNA and non-viral gene delivery vector poly (ethyleneimine)-hyaluronic acid within the core and sheath polymer of poly(ethylene) glycol and poly(ε-caprolactone), respectively. The cumulative releases of plasmid DNA and the non-viral gene delivery vector from the core and sheath of fiber mesh scaffolds over a time period of almost 2 months showed a dramatic increase in transfection efficiency compared to the control group [108]. Though variously modified nanofiber scaffolds are used today to protect and encapsulate plasmid DNA-like genetic materials, the blending of plasmid DNA with an electrospun solution does not facilitate proper encapsulation and protection of biotherapeutics. In this case, the incorporated plasmid DNA is not uniformly distributed throughout the nanofiber scaffolds, which may hamper their release profile. Hence, surface modification with a cationic polymer could solve these issues. Kim et al. designed DNA-loaded surface modified nanofibers for epidermal gene delivery with a matrix metalloproteinases (MMPs) responsive control release behavior. Here, a MMPs-cleavable linker was used for surface modification of as-synthesized poly(ethylene) glycol/poly(ε-caprolactone) nanofiber meshes with linear polyethyleneimine (LPEI) so that the external MMPs can breakdown the MMPs-linker and facilitate the MMPs-responsive control release of the DNA-loaded polymer [119]. The release studies revealed that more than 80% DNA and almost 80% LPEI can release from the proposed nanofiber mashes over a 72 h time period whereas the transfection efficiency mainly depended on the charge ratio between DNA and LPEI rather than the amount of release.

To date, various natural and synthetic polymers have been used to fabricate electrospun nanofibers in order to allow the successful delivery of genetic materials to the target site. In addition, the hybrid blending of natural and synthetic polymers has also been introduced for the same purpose. In Table 1 we illustrate the usages, designs and advances of various nanofibers as delivery platforms for DNA and RNA, respectively.




#### **Table 1.** *Cont.*


**Table 1.** *Cont.*

pDNA: plasmid DNA, PEG: polyethylene glycol, PLA: poly(lactide), PDLLA: poly(D,L-lactide), PLGA: poly(lactide-co-glycolide), PEI: poly (ethyleneimine), HA: hyaluronic acid, PCL: poly(caprolactone), PECL: poly(ε-caprolactone), PELA: poly(DL-lactide)–poly(ethylene glycol), Hap: hydroxyapetite, P4VP: poly(4-vinyl pyridine), LPEI: linear polyethyleneimine, ELP: elastin-like polypeptides, siRNA: short interfering RNA, PEG: polyethylene glycol, PLGA: poly(lactide-co-glycolide), PCL: poly(caprolactone), PECL: poly(ε-caprolactone), LPEI: linear polyethyleneimine, PCLEEP: poly(ε-caprolactone-co-ethyl ethylene phosphate), ZnGa2O4:Cr: chromium-doped zinc gallate.

Nanofiber-based siRNA delivery is also in the spotlight due to its promising mechanism for silencing specific gene expression, targeting various diseases. Those genes have the ability to develop genetic mutations and block the secretion of inhibitory factors resulting in excess cell proliferation that may lead to cancer. To date, various electrospun nanofiber scaffolds have been used as a siRNA nanocarrier to deliver siRNA into the physiological system. Among them, PCL is the most used nanofiber scaffold that facilitates high siRNA loading efficiency, local delivery of siRNA, control release behavior, higher cellular transfection, manageable toxicity and maximum gene silencing properties [114].

However, the hydrophobic nature and slow degradation properties of PCL are responsible for the slow release properties of encapsulated siRNA [110,121]. To overcome this issue, surface modification, the formation of the block polymer and the polydopamine coating of PCL types strategies are taken into consideration which are briefly described in Table 1. Peptide-based nanofiber scaffolds have also been introduced as an siRNA nanocarrier targeting neurodegenerative disease. Their unique characteristics facilitate targetwise siRNA release and accumulation, a high residence period of siRNA in the brain region, a promising gene silencing profile, and ensure genetic intervention [123]. In addition, for the first time, scientists proposed a zein nanofiber-inspired siRNA delivery system that ensures proper siRNA encapsulation and the sufficient release of siRNA, high loading efficiency, cellular attachment, and transfection of siRNA. This zein-based electrospun nanofiber successfully preserves the efficiency of siRNA [113]. Various nanoparticles, except for PCL, such as PEG (polyethylene glycol), PCLEEP (poly(ε-caprolactone-co-ethyl ethylene phosphate)), ZnGa2O4:Cr (chromium-doped zinc gallate), P-G3A3KRK (Palmitoyl-GGGAAAKRK peptide), Zein, PECL (poly(ε-caprolactone)), PEG-b-P4VP ((Poly(ethylene glycol)-*b*-poly(4-vinylpyridine)), PLGA (poly(lactide-co-glycolide)), LPEI (linear polyethyleneimine), ELP (elastin-like polypeptides), PDLLA (poly(D,L-lactide)), and many more are used as a nucleic acid carrier [111–113,120,122,123,125] and are summarized in Table 1.
