Nanofiber as a novel vehicle for transdermal delivery of therapeutic agents: challenges and opportunities

Transdermal delivery of drugs is a quite challenging task for pharmaceutical scientists. The transdermal route is preferred over the oral route due to various advantages like avoidance of the first-pass effect, non-invasiveness, and high patient compliance. Therefore, it is necessary to develop an effective carrier system that enables the effective passage of the drug through the dermal barrier. Various novel drug delivery systems are used to enhance the permeation of a variety of drugs through the skin barrier. Researchers around the globe have explored nanofibers for the transdermal delivery of various therapeutic agents. Nanofibers are designed to have a high concentration of therapeutic agents in them promoting their flux through various skin layers. Polymeric nanofibers can be explored for the loading of both hydrophilic and lipophilic drugs. Biopolymer-based nanofibers have been also explored for transdermal delivery. They are capable of controlling the release of therapeutic agents for a prolonged time. The literature presented in this review paper provides significant proof that nanofibers will have an intense impact on the transdermal delivery of different bioactive molecules in the future.


Background
Skin is the outermost lipidic barrier of the body with a thickness of 20-25 µm [1]. Besides the barrier function, it also helps in the absorption of various therapeutic and non-therapeutic molecules [2]. The presence of skin appendages like hair follicles can also be responsible for the passive absorption of drug molecules through the transdermal route ( Fig. 1) [3]. Since drug molecules can directly enter into the systemic circulation after crossing this barrier, therefore, this route has attracted pharmaceutical scientists to perform research in the field of drug delivery for the last two decades [4]. The transdermal route is considered a better alternative to the oral route of drugs due to the prevention of dose fluctuations, first-pass hepatic metabolism, and increased bioavailability [5]. Moreover, the non-invasive nature and ease of application of dosage form through this route have helped to gain popularity among patients [6]. Numerous factors should be considered before developing transdermal delivery systems of drugs like skin barrier only allows penetration of hydrophobic drug molecules through it with molecular weight less than 500 kDa (kilodaltons) [7]. The rate of influx of drugs is very slow through this barrier. However, the effective delivery of hydrophilic drugs through the skin is still a challenging task [8].
There are various nanocarrier systems like liposomes, niosomes, solid lipid nanoparticles, nanostructured lipid carriers, ethosomes, and polymeric nanoparticles which are explored for effective transdermal delivery of drugs [9]. However, polymeric micro or nanofibers have gained special attention for effective transdermal delivery of drugs for the last decade [10]. Various methods explored for the production of nanofibers are electrospinning, template synthesis, and phase separation [11]. However, electrospinning is the most widely used technique among all of them due to its cost-effectiveness and simplicity [12]. Nanofibers are generated in the form of the mat from electrospinning (Fig. 2) revealing various advantages like high surface area, nanopore size, and unique physicochemical properties [13]. These characteristic traits of nanofibers make them a suitable candidate for the delivery of drugs and genes [14]. Nanofibers may be an excellent choice for tissue engineering and dressing wounds due to their capability to produce a local effect [15]. There are various categories of drugs like anticancer, NSAIDs (Non-steroidal anti-inflammatory drugs), and antibiotics which are delivered through the transdermal route exploring nanofibrous mats [16]. This paper summarizes the utility of nanofibrous mats/scaffolds for transdermal delivery of various categories of bioactive molecules.

Methods of production of nanofibers
Nanofibers come under the category of nanostructured vehicles having a diameter of individual fiber below 100 nm [17]. Although developed fibers with a diameter in the range of 100-1000 nm are also designated as nanofibers and they are generally manufactured using a technique known as electrospinning [18]. Various methods explored for the production of nanofibers are shown in Fig. 3.

Self-assembly method
There is a spontaneous arrangement of atomic/molecular aggregates into structurally defined nanofibrous form in this method. This method leads to the production of nanofibers of a size range up to 100 nm. This method requires a higher time to generate nanofibers, therefore, less commonly implemented. However, nanofibers manufactured through a self-assembly method can mimic natural materials like chitin (polysaccharide) very closely that has been explored in tissue engineering [19].

Template synthesis method
Template synthesis involves the use of nanoporous membranes that are available in the form of templates to extrude available fibers of different sizes into the nanoscale size range. The size of nanofibers produced lies in the range of 200-400 nm [20].

Phase-separation method
This method involves lyophilization of polymeric blend resulting in the formation of the nanofibrous mat. However, this method is very time-consuming and nanofibers obtained through this method are shorter in length with a size range of 50-500 nm [21].

Melt-blown technology
Melt blown method involves extrusion of polymer blend across a minute orifice followed by passage through heated air stream with a very high velocity. The size of nanofibers produced exploring this method is 150-1000 nm [22].

Electrospinning
Electrospinning is the most widely used technique for nanofiber production. Fibers generated through the electrospinning method may lie in the nanometer to the micrometer size range. It is considered a cheap and scalable technique for the production of nanofibers [23]. Nanofibers are also produced by a modified electrospinning technique known as 'nanospider' . This technique generates nanofibers in the form of nonwovens with a diameter range of 50-300 nm [24]. Nanofibrous nonwovens are widely explored in various fields of biomedical engineering like wound dressing and tissue engineering, transdermal drug delivery, and enzyme immobilization [25]. Electrospinning involves the preparation of polymeric melt/solution initially followed by application of electric charge on it after its extrusion from nozzle/ syringe/pipette [26]. Finally, the developed nanofibers are collected on the aluminum wall due to electrostatic attraction between polymer and wall (due to the presence of opposite charge on both) (Fig. 4) [27].

Methodologies of drug loading in nanofibers
Various methodologies of loading drugs into nanofibers are discussed below:

Co-electrospinning
This approach involves the simple mixing of the polymeric solution with the drug before the initiation of electrospinning. A homogeneous solution of drug and polymer in a single solvent is further subjected to electrospinning and this type of electrospinning is called co-electrospinning [28]. This technique shows high loading efficacy and homogeneous drug distribution within the nanofibrous network [29]. The loading efficiency of nanofibers produced through this method depends on the physicochemical properties of the polymer used followed by the interaction of polymers with drug molecules [30]. The morphology of nanofibers and the distribution of drug molecules within them may affect their release kinetics [31]. Various natural polymers like gelatin, collagen, and chitosan are used to develop nanofibers loaded with hydrophilic drugs due to their complete dissolution in the aqueous phase [32]. Nanofibers produced through this method collapse during the cross-linking process creating problems in the electrospinning process. This can be due to the reduced viscosity of the solution and this problem can be overcome by using synthetic hydrophilic polymers like PEO (Polyethylene oxide) additionally. Nanofibers developed through this method can lead to a burst release effect also [33].

Immobilization of drug molecules on the surface of nanofibers
Various therapeutic drug molecules can be loaded in nanofibers following the surface immobilization method through various physical and chemical mechanisms. Various forces involved in physical immobilization are electrostatic forces, hydrogen bonding, or weak van der Waals forces [34]. Chemical immobilization involves the direct attachment of drug molecules over the nanofiber surface through functionalization with various groups like thiol, carboxyl, hydroxyl, and amine [35]. The surface immobilization method does not cause denaturation of drug molecules as observed in the case of the co-electrospinning method due to excessive use of organic solvents and high voltage [36]. The amount of drug to be immobilized on the surface of nanofibers can also be controlled by using this technique through optimization of drug feeding ratio. This approach is also capable of blocking initial burst release from nanofibers promoting slow release kinetics [37].

Co-axial electrospinning
Immiscibility between drug molecules and the polymer may create problems in the co-electrospinning process. Therefore, for loading different kinds of drugs having a difference in solubilities in polymers a new technique named 'co-axial electrospinning' is used [38]. Co-axial electrospinning is done with the help of a spinneret needle having one inner and one outer nozzle organized concentrically. There is the presence of two different chambers for the handling of sheath solution and core solution. The final solution is ejected from the co-axial cone (Fig. 5) [39]. This technique enables the electrospinning of two non-miscible polymers having therapeutic agents in core and sheath as well [40]. Electrospinning through this technique results in high drug loading capacity and prevention of initial burst release due to the presence of a stagnant sheath [41]. Generally, hydrophilic polymers and therapeutic agents like proteins are enclosed in the core portion while hydrophilic elements remain in the sheath. Co-axial electrospinning requires controlling a large number of factors like the feeding speed of polymeric solution, voltage application, and concentration of therapeutic agents for the production of nanofibers with proper core and sheath structure [42].

Emulsion electrospinning
Emulsion electrospinning involves the emulsification of an aqueous solution of a therapeutic agent or protein with a lipophilic polymeric solution [43]. Furthermore, the drug-loaded phase is disseminated in the nanofibers at the termination of electrospinning (Fig. 6). While using this method, the distribution of drug molecules within the nanofiber is totally dependant on the ratio of hydrophilic to the lipophilic solution used [44]. Therapeutic agents and polymers can be dissolved in suitable solvents using this technique. This method involves minimal exposure to the therapeutic agent with an organic solvent [45]. The emulsion electrospinning method allows the use of a variety of hydrophilic drugs and lipophilic polymer combinations [46]. The existence of interfacial tension and strong shearing forces between two phases of the emulsion can degrade the proteinaceous drug molecules due to their high sensitivity [47]. The use of ultrasonication methodology in this electrospinning technique can damage the drug molecules reducing the efficacy of nanofiber produced [48].

Applications of nanofibers in transdermal delivery of various therapeutic agents
Various categories of drugs that are delivered through a transdermal route using nanofibers are discussed below:

Antibiotics or antimicrobial drugs
Cutaneous wounds infection may be responsible for increased healing duration, a longer period of hospitalization, and death of the patients many times [14]. Skin infections can be effectively treated by using antibiotics/antimicrobial drugs locally. Pharmaceutical scientists have investigated various antibiotics/antimicrobial drugs impregnated into nanofibers for the treatment of cutaneous wounds [16]. Kataria et al. [49] investigated ciprofloxacin-loaded polyvinyl alcohol and sodium alginate-based nanofibers for localized delivery and to treat the wound in rabbits. Ciprofloxacin-loaded nanofibers showed drug release in-vitro following Higuchi and Korsmeyer-Peppas model. The wound healing capacity of nanofibers was determined using hydroxyproline assay in wounds. Ciprofloxacin-loaded nanofibers showed the highest amount of hydroxyproline (8.39/100 mg of wound bed) in the animal wound after twenty days compared to the marketed formulation of ciprofloxacin (7.91/100 mg of wound bed) indicating their high effectiveness [49]. Furthermore, nanofibers composed of polymers poly(vinyl alcohol) and lysine and impregnated with ibuprofen (an anti-inflammatory agent) and lavender oil (anti-bacterial agent) were investigated by Sequeira et al. [50] for the acceleration of the wound healing process. Ibuprofen was loaded using the co-electrospinning technique while lavender oil was loaded using the surface adsorption technique in nanofibers. Nanofibers loaded with ibuprofen displayed a reduction in the time scale of the wound healing inflammatory phase. However, lavender oil-loaded nanofibers showed a very high in-vitro antibacterial efficacy against S. aureus and P. aeruginosa compared to nanofibers loaded with ibuprofen without affecting dermal fibroblasts [50] (Fig. 7). Later on, Iqbal et al. [51] determined the efficacy of chitosan/poly(vinyl alcohol) nanofibers loaded with cefadroxil monohydrate against resistant gram-positive bacteria S. aureus responsible for chronic skin fungal infection. Nanofibers with 30:70 of chitosan/ poly(vinyl alcohol) were considered as optimized and these developed nanofibers showed high in-vitro antimicrobial activity against resistant S. aureus followed by low toxicity towards epidermal keratinocytes as depicted in MTT assay. They were considered a better alternative for the treatment of chronic skin fungal infections [51]. Table 1 gives a brief overview of nanofibers for transdermal delivery of various antimicrobial/antibiotic drugs.

Antifungal drugs
Polymeric electrospun nanofibers are also explored for transdermal delivery of various antifungal drugs. Harini et al. [61] investigated the antifungal potential of polycaprolactone (PCL)/egg lecithin-based nanofibers impregnated with terbinafine hydrochloride to treat skin fungal infections. Developed nanofibers with diameter 127.7 ± 43.7 nm were found non-cytotoxic towards human dermal fibroblasts as revealed through confocal microscopy and they also showed excellent in-vitro antifungal activity against different fungal strains like Epidermophyton and Trichophyton mentagrophytes responsible for topical fungal infections [61]. Furthermore, Paskiabi et al. [62] formulated nanofibers loaded with terbinafine hydrochloride (TFH) using polymers polycaprolactone (PCL) and gelatin (50:50 w/w) using glutaraldehyde (GTA) as a cross-linking agent. TBHloaded nanofibers showed non-cytotoxic behavior as evaluated in L929 cells. Cross-linked nanofibers loaded with TBH showed 100% drug loading followed by a high in-vitro antifungal activity against T. mentagrophytes and A. fumigates and less effective against C. albicans (Fig. 8) [62]. Later on, voriconazole impregnated polyvinyl alcohol (PVA)/sodium alginate nanofibers were formulated by Esentürk et al. [63] and further cross-linked with glutaraldehyde (GTA) for effective delivery through the transdermal route. Cross-linked polymer composite nanofibers loaded with voriconazole showed high drug loading (96.45 ± 5.91%) followed by low in-vitro cytotoxicity against mouse fibroblast cells. Voriconazole impregnated polyvinyl alcohol (PVA)/sodium alginate nanofibers showed high in-vitro antifungal activity against C. albicans and deeper penetration of drug in the lower skin layer compared to the solution of voriconazole in propylene glycol [63]. Esenturk et al. [64] explored polyurethane/polyvinylpyrrolidone/silk nanofibrous mats loaded with sertaconazole nitrate for transdermal treatment of fungal infection caused by C. albicans. Developed nanofibers showed approximately 89.97 ± 1.40% loading of sertaconazole nitrate and sustained its release for up to 168 h in-vitro. Sertaconazole nitrate loaded in nanofibers showed fungistatic action towards C. albicans and excellent in-vitro biocompatibility for mouse fibroblast cell lines as revealed in the CCK-8 assay [64].
Later on, Azarbayjani et al. [65] developed nanofibers of Polyvinyl alcohol and chitosan for transdermal  delivery of hydroquinone and investigated the effect of chitosan on their efficacy. Optimized hydroquinoneloaded nanofibers showed a diameter of 537.24 ± 52.5 nm and drug loading of 4.4%. Increasing the concentration of chitosan up to 2% in the formulation did not cause any significant changes in nanofiber diameter, loading percentage, and in-vitro antifungal activity against Candida albicans, however, it was able to increase the in-vitro release of hydroquinone at 32 °C compared to 25 °C [65].

Anti-inflammatory drugs
Electrospun nanofibers have also been investigated by pharmaceutical scientists for the transdermal delivery of many anti-inflammatory drugs. Shi et al. [66] investigated Cellulose acetate/poly(vinyl pyrrolidone) based nanofibers impregnated with ibuprofen for transdermal delivery.
Optimized nanofibers showed a diameter of 167 ± 88 nm and X-Ray Diffraction analysis of nanofibers revealed uniform distribution of ibuprofen in the nanofibrous network in amorphous form. Developed nanofibers showed better in-vitro skin permeation of the drug followed by increased water vapor permeability compared to the conventional transdermal patch of the same drug indicating their high thermodynamic stability [66]. Furthermore, rosmarinic acid (RosA) loaded cellulose acetate (CA) nanofibers were evaluated by Vatankhah [67] for in-vitro anti-inflammatory activity (determination through protein denaturation assay), cytotoxicity, and antioxidant effect. Nanofibers formulated using 10% rosmarinic acid were considered as optimized and they showed diameter 331 ± 85 nm and drug loading (%) 84 ± 4%. These nanofibers were capable of extending the release of rosmarinic acid up to 64 h through the Fickian diffusion mechanism and higher in-vitro anti-inflammatory activity compared to the ibuprofen solution. A promising  in-vitro antioxidant effect was observed for nanofibers followed by very low cytotoxicity in epithelial cells ( Fig. 9) [67]. Later on, an evaluation of poly(vinyl alcohol) based nanofibers loaded with diclofenac enclosed in zein nanoparticles was carried out by Ghalei et al. [68]. Developed nanofibers showed a diameter of 324.42 ± 72.80 nm and good tensile properties for topical application. Nanofibers containing diclofenac loaded inside zein nanoparticles were considered best for wound healing due to their better in-vitro attachment in fibroblasts followed by the promotion of their proliferation [68]. The utility of nanofibers for transdermal delivery of various antiinflammatory drugs is given below in Table 2.

Anticancer drugs
The local effect of anticancer drugs in the skin can be improved by loading them into a nanofibrous mat. Rengifo et al. [74] developed pyrazoline H3TM04 loaded nanoparticles and further impregnated them into nanofibers composed of polyethylene oxide-chitosan for  the treatment of skin cancer. Optimized nanoparticles loaded nanofibers showed a diameter of 197.8 ± 4.1 nm and uniform distribution of nanoparticles throughout the nanofiber matrix followed by the extended-release of pyrazoline H3TM04 up to 120 h. Developed nanofibers also enhanced in-vitro transport pyrazoline H3TM04 across the epidermal skin layer followed by excellent invitro cytotoxicity against B16F10 melanoma cells [74]. Furthermore, molybdenum oxide-loaded nanoparticles were prepared by Janani et al. [75] and impregnated into polycaprolactone (PCL) nanofibers for evaluation of their skin anticancer potential in zebrafish. Nanofibrous mat loaded with molybdenum oxide nanoparticles showed an average diameter of 200 nm and a significant reduction in in-vitro cell viability (> 50%) in A431 cells through mitochondrial dependant apoptosis. Nanofibers loaded with molybdenum oxide nanoparticles showed reduced skin cancer progression in zebrafish by more than 30% within two weeks (Fig. 10) [75]. Table 3 discloses the role of polymeric nanofibers in the transdermal delivery of various antineoplastic drugs.

Other categories of drugs
There are other categories of drugs other than those discussed above which can be delivered through the transdermal route exploring nanofibers for producing the evident pharmacological effect. Madhaiyan et al. [81] investigated polycaprolactone polymer-based nanofibers loaded with Vitamin B 12 for effective delivery through the transdermal route. Vitamin B 12 loaded nanofibers showed an average diameter of 1.226 ± 0.108 µm and 89% drug loading capacity followed by high mechanical strength and excellent surface wettability. Surface treatment of Vitamin B 12 loaded nanofibers with plasma greatly affected in-vitro release ate of Vitamin B 12 from nanofibers. Nanofibers treated with plasma for 60 s showed the highest release of Vitamin B 12 within 50 h (Fig. 11). This could be due to the increased hydrophilicity of the nanofiber membrane after treatment with plasma [81]. Furthermore, hydrocortisone-loaded polyacrylonitrilebased nanofibers were formulated by Hemati Azandaryani et al. [82] and were investigated for topical treatment of psoriasis by varying amounts of surfactant Tween 80 in nanofiber composition. Nanofibers produced using polyacrylonitrile polymer along with 5% Tween 80 surfactant showed the lowest diameter (160.11 ± 30.11 nm) and maximum tensile strength (15.35 MPa) followed by the highest in-vitro drug release for 12 h and minimum cytotoxic effect against HUVEC cell lines indicating their efficacy in transdermal drug delivery for the treatment of psoriasis [82].
The role of polymeric nanofibers in transdermal drug delivery of various therapeutic agents is given in Table 4.

Biopolymer based nanofibers in transdermal delivery
Biopolymers are polymeric materials that are manufactured from natural provenance. Biopolymers are chemically produced from biological materials or their complete biosynthesis can be done by living organisms [90]. Various examples of biopolymers are cellulose, chitosan, hemicellulose, silk, and lignin. These biopolymers may be biocompatible and biodegradable promoting their use in drug delivery [91]. Nanomaterials that are derived usually derived from cellulose are called nanocellulose materials. These materials can be classified into three categories namely nanofibrillated cellulose, bacterial nanocellulose, and nanocrystalline cellulose     [92]. nanocellulose based materials show elevated surface area, ease of chemical modification, and a higher value of specific strength. Hence, nanocellulose can be explored as a good candidate for various biomedical utilities [93]. El-Wakil et al. [94] investigated the wound healing potential of coffee extract impregnated into bacterial cellulose (produced from kombucha tea fungus) biocomposites. Biocomposites composed of minimum coffee extract and cellulose amount disclosed maximum tensile strength (3.35 MPa) and transmission of water vapors (3184.94 ± 198.07 g/m 2 /day) followed by least release of polyphenols in-vitro in PBS (pH 7.4) considered suitable for wound healing [94]. Furthermore, Shan et al. [95] developed cellulose nanocrystal incorporated calcium cross-linked sodium alginate/gelatin nanofibers for efficient wound healing. Developed nanofibers showed in-vitro non-toxicity against mouse embryonic fibroblast and improved cell adhesion. The cellulose nanocrystal incorporated calcium cross-linked sodium alginate/gelatin nanofibers showed excellent wound healing in Sprague Dawley rats through a re-epithelialization mechanism compared to the control group [95].

Description of patents related to the use of nanofibers for transdermal delivery of various therapeutic agents
A detailed literature investigation revealed the excellent therapeutic potential of nanofibers to treat various abnormal conditions of the skin. These nanofibrous scaffolds can be explored as a better alternative to conventional drug delivery systems for the transdermal treatment of various skin disorders. Hence, pharmaceutical researchers are filing patents regarding the use of nanofibers for transdermal drug delivery of various therapeutic agents. Table 5 discloses the list of patents granted regarding this context.

Limitations and challenges in the exploration of nanofibers for transdermal drug delivery
Polymeric nanofibers have shown promising potential in transdermal drug delivery, however, many significant challenges must be taken into consideration. All the research investigations available in the literature describe either in-vitro or in-vivo (in different animal models) efficacy of transdermal nanofibers. However, the clinical efficacy determination of nanofibers explored through the transdermal route is still a challenge. Clinical evaluation of nanofibers will be exorbitant and laborious. It will require high speculation by the industries or government funding agencies of the countries. The second major concern will be regarding the scale-up of transdermal nanofibers. Nanofibers are effectively produced through the electrospinning process following a low flow rate of polymeric solution, making the production process more time-consuming. Nanofibers production is also affected by humidity, hence it might be a challenging factor for bulk processing and scale-up of nanofibers. Furthermore, the production of transdermal nanofibers with GMP (Good manufacturing practices) standards will be required. The development of standard and universally accepted electrospinning protocol will govern their quick entrance into the pharmaceutical market.

Conclusions
Nanofibers have been explored for transdermal drug delivery due to their various merits like high drug loading, surface-to-volume ratio, and similarity with the extracellular matrix. Successful production of the nanofibrous mat is dependent on appropriate polymers and solvent selection for electrospinning. A nanofiber suitable for transdermal drug delivery can be produced using multiple polymer blends for electrospinning. Polymeric nanofibrous mat loaded with a therapeutic agent has the caliber to control/prolong its release transdermally.
Transdermal nanofibers have shown their therapeutic potential in various preclinical investigations carried out by various pharmaceutical scientists. However, their entrance into the pharmaceutical market will be governed by developing effective scale-up technologies and detailed clinical evaluation.