Blend Electrospinning, Coaxial Electrospinning, and Emulsion 11 Electrospinning Techniques

Blend electrospinning, coaxial electrospinning, and emulsion 11 electrospinning techniques

Matej Buzgo1, Andrea Mickova1, Michala Rampichova2 and Miroslav Doupnik1 1InoCure s.r.o., Klimentska´ 1652/36, 110 00 Prague, Czech Republic, 2Laboratory of Tissue Engineering, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, v.v.i, Vı´denskaˇ ´ 1083,142 20, Prague 4, Czech Republic

11.1 Advanced electrospinning techniques

Electrospinning is a recent fiber-forming technology enabling production of fibrous scaffolds for use in tissue engineering [1,2], biomedicine [3,4], filtration [5]¸ and other technical applications [6,7]. The process is based on drawing of fibers from polymeric solution or melt using high electrostatic forces. Apparatus for electrospinning uses high-voltage power supplies to generate potential up to 50 kV. Upon effect of electrostatic forces, the polymeric solution is attracted towards the opposite electrode and a straight jet of solution is formed from a conical protrusion, often called a Taylor cone, leading to fibers with diameter in submicron range [8]. Classical electrospinning process is used for the formation of porous matrices, which was reviewed in numerous publications [1,2,9,10]. The key properties of fibrous meshes are high porosity, very high surface-to-volume ratio, high pore interconnectivity, and thin fiber diameter [1,2]. The morphology of fibrous layer is mim- icking the structure of extracellular matrix and facilitates application of electrospun scaffolds is in the field of tissue engineering and nanomedicine. Besides medical field, the structure of electrospun fibrous meshes has advantageous properties for application in liquid and air filtration [5]. Apart from classical electrospinning techniques, advanced electrospinning techniques attracted huge attention due to possibility to prepare multimaterial and drug functionalized materials. This chapter focuses on properties and use of these advanced methods and strategies.

11.2 Nanofibers as a drug delivery system

The ability to regulated drug delivery is changing the way of how drugs are admin- istrated to patients. In classical dosage formulation, the drugs are delivered alone or

Core-Shell Nanostructures for Drug Delivery and Theranostics. DOI: https://doi.org/10.1016/B978-0-08-102198-9.00011-9 © 2018 Elsevier Ltd. All rights reserved. 326 Core-Shell Nanostructures for Drug Delivery and Theranostics in combination with excipients in single doses. They have rapid bioavailability but also rapid clearance times. This dosage mode is ideal for drugs with desired rapid action (i.e., antiinflammatory drugs, pain reducing drugs, antibiotics); however, for long-term and chronic application, they are associated with the need for periodic dosage intervals. Drug delivery systems prolong this time and enable more convenient dosage to patients. Nevertheless, combination with drug delivery system enables elimination of drug degradation (i.e., first pass metabolism in liver), elimi- nate systemic effect of drugs (i.e., toxicity of cytostatics), and target drug release to desired areas (i.e., tumor delivery) [11,12]. Electrospun nanofibers present numerous advantages for their use as drug delivery systems [1,9]. Due to their enormous specific surface area, they enable adsorption of drugs and simple functionalization. In addition, the encapsulated drugs are efficiently released from bulk matrix of fibers through the high surface depending on diffusion/degradation mechanisms. The process enables regulation of release depending on fiber chemical and morphological composition [13]. Due to pore interconnectivity and high porosity, the drugs could freely diffuse from the mesh after release. The scaffold structure in the form of fibrous mesh facilitates the use of advanced nanofibers as topical or implantable depot release devices. In contrary to nano and microparticles, the nanofibrous systems are less suited for injection delivery and release in systemic circulation. However, the combination of biomi- micking surface and drug release properties allows utilization of such systems in tissue engineering (i.e., implants and scaffolds) [14 18], as patches (i.e., skin delivery, buccal delivery, implantable hernia, or cardiac patches) [19 21] and dressings (i.e., wound dressings) [19,21]. Electrospun scaffolds could be functionalized by numerous methods. Drugs can either be attached superficially to the nanofiber surface or internalized (encapsulated) into the nanofiber core. Drug adsorption is the simplest method. The loading amount and adsorption/desorption rate is dependent on surface properties of fibers and finding optimal composition is often problematic. Drug encapsulation aims to diminish this major shortcoming. There are several ways to encapsulate drugs into the nanofiber matrix by electrospinning. The most common methods include blend electrospinning, emulsion electrospinning, and coaxial electrospinning.

11.3 Functionalization of nanofibers by surface adsorption

Physical adsorption to the nanofiber surface is a simple way to functionalize nanofibers for drug delivery. The large specific surface of nanofibers means that they can be loaded with a high amount of drugs. However, the adsorbed molecules are often released too rapidly and the systems are more suitable for short-term drug delivery. The adsorption/desorption ratio is regulated by surface properties of electrospun fibers. For instance, cationic exchange surface was developed using sulfonated polystyrene fibers [22]. The fibers were used for binding of five cationic drugs Blend electrospinning, coaxial electrospinning, and emulsion electrospinning techniques 327

(dextromethorphan, chlorpheniramine, diphenhydramine, propranolol, and salbutamol). The drug adsorption/desorption behavior correlated with drug properties (pKa, lipophilicity, molecule size, and steric properties) and solvent properties (concentration and valence of ions in releasing solution). The salbutamol was released with the fastest rate (order of minutes) due to its lowest molecular weight, lowest hydrophobicity (low hydrophobic interactions), and lower affinity of cationic groups to sulfonylated surface. With the increase of these parameters, the desorption rate was slower (order of hours). The results illustrate the need of complex optimization of numerous parameters to find proper balance between drug adsorption/desorption rate and optimal delivery time interval for molecules bound on the surface of fibers. Electrospun fibers were combined with a range of drugs by surface adsorption (i.e., tetracycline, ciprofloxacin, and bisphenol) [23,24]. In addition, proteins were also loaded on the surface of fibers. However, susceptible molecules are not protected from environmental degradation and their bioactivity is lowered. Nie et al. [25] showed that immobilization of BMP-2 to the surface of nanofibers resulted in 75% release during the first 5 days. Similarly, other growth factors, such as epidermal growth factors (EGF) [26], basic fibroblast growth factor (bFGF) [27], nerve growth factor (NGF), and ciliary neurotrophic factor [28], were bound on the surface of fibers. To increase the loading capacity of proteins, specific motives have been imple- mented to the surface of fibers. Lam et al. [27]. prepared EGF and bFGF functionalized fibers by coating of fiber surface by heparin. The factors interacted with heparin moieties and helped in improving of axon growth on scaffolds. Similarly, PVA fibers with phosphatidylcholine on surface enabled enhanced adsorption of insulin and stimulation of cartilage defect restoration on model of minipig [29]. Nevertheless, the nanofibers could also be further functionalized by surface- bound drug delivery systems. Nanofibers, due to their enormous surface, are serving as a platform for binding of such systems, enable localization of drug release to desired areas of scaffolds, and provide spatial localization on site of implantation. The surface of fibers was modified by binding liposomes as delivery system for proteins. Rampichova et al. [30] adsorbed liposomes with encapsulate fetal bovine serum for stimulation of chondrocyte proliferation. Similar system was utilized for release of gentamicin from liposomes [31]. Nevertheless, other membrane systems were used as drug delivery systems. Among them, Vocetkova et al. [32] and Jakubova et al. [33] used polycaprolactone (PCL) nanofibers as a scaffold for adhesion of human platelets. Platelets serve as natural source of growth factors and stim- ulated fibroblasts, keratinocytes, melanocytes, and chondrocytes. Nanofibers were combined also with nano/microparticles particles for the development of composite systems [34]. For instance, nanofibers were coated with silica nanoparticles containing ibuprofen [35]. Coating by adsorptive polymer (polydopamine) facilitates improved adhesion of nanoparticles and enables further regulation of additional drugs encapsulated in fibers (i.e., doxorubicin). Such multistage release systems have potential in medication of complex diseases. Protein-loaded microspheres were electrosprayed on poly(lactic-co-glycolic acid) (PLGA) fibers to prepare drug releasing scaffolds [36,37]. The system enabled to prepare diverse density of 328 Core-Shell Nanostructures for Drug Delivery and Theranostics electrosprayed particles resulting in the formation of gradient of released biomolecules (BMP-2). Bock et al. [38] prepared protein releasing composite by combining electrospinning and electrospraying. The system was able to release proteins for 18 days. In addition, osteoinductive coating were prepared by electrospraying of hydroxyapatite nanoparticles on electrospun polyhydroxybutyrate (PHB) scaffolds [39] and polycaprolactone-co-lactic acid (PLCL) scaffolds [40].

11.4 Blend electrospinning (co-electrospinning)

Blend electrospinning is based on mixing of a drug with polymeric solution prior to electrospinning process. The drug is dissolved or dispersed in the polymeric matrix, and upon favorable conditions, the process results in encapsulated drug with prolonged release profile. The release from polymeric matrices is governed either by desorption/diffusion or dissolution/erosion of polymeric matrix [41]. The release from nonbiodegradable polymers is governed by diffusion of drug through the polymeric layer. In case of biodegradable polymers, an additional factor, related to decomposition (dissolution/erosion) of the system, must be considered. If the solvent enters the interior of polymeric matrix, swelling associated with increase of volume and rearrangement of polymeric chains occurs (e.g., hydrophilic matrices in polar solvents). Thus generally the release is either diffusion or erosion controlled [41]. In case of diffusion controlled release, the solvent enters the polymeric matrix and results in swelling polymeric matrix. If the solvent supports the dissolution of drug (solid to liquid), then the release is connected with diffusion through the polymer/solvent layer. The diffusion is driven by concentration sink from inside of polymeric matrix to the outside. If the release of drug is controlled solely by diffusion through the matrix, it corresponds to Fickian type (Type I) diffusion [41,42]. In case of polymer swelling and changes associated with polymer matrix rearrangement, the diffusion type corresponds to polymer swelling diffusion mechanism (Type II) [41,42]. In addition, non-Fickian diffusion (Type III) depends both on polymer swelling and drug diffusion [41,42]. On the other hand, the release may be controlled strictly by the degradation of polymeric matrix. The release mechanism associated with such systems is polymer erosion/degradation mechanism (Supra Type II) [41,42]. Blend electrospinning has been tested for a wide range of substances such as antibiotics [43 48], cytostatic [49 52], and antiinflammatory drugs [53 55]. The technology is especially useful for the delivery of small molecules. Blend electrospinning was successfully used for encapsulation of antimicrobial peptides [56 58]. Utilization of blend electrospinning for delivery of proteins is problematic due to harsh environment during encapsulation. Unfortunately, most biocompatible polymers (e.g., PCL, PLGA, PU, etc.) are soluble in organic solvents. Incubation of proteins in such solutions results in unfolding and changes in protein conformation. As a result, the process is often associated with decreased bioactivity of delivered protein-based therapeutics [59]. On the other hand, polar polymers have often rapid Blend electrospinning, coaxial electrospinning, and emulsion electrospinning techniques 329 solubility and the release times are rapid. One possibility is the regulation of nanofiber stability by cross-linking. Buzgo et al. [13] prepared polyvinyl alcohol (PVA) nanofibers stabilized by various amount of PEG-based cross-linker. The variation in cross-linking ratio enables regulation of time-release of model active molecules. Examples of using polar polymers for delivery of proteins include silk fibroin nanofibers with BMP-2 for stimulation of bone cells [52].

11.5 Coaxial electrospinning

Coaxial electrospinning is already well established as a method for producing drug- releasing nanofibers. Coaxial electrospinning is based on simultaneous co-spinning two polymeric liquids. The general system for core/shell electrospinning is based on utilization of two needles, which are coaxially placed together. The spinneret therefore enables the formation of composite polymeric droplet—the inner (core) liquid is pumped through internal needle, and the shell material is delivered through the outer needle. Upon application of strong electric field, the polymeric droplet results in the formation of composite electrospinning jets and the formation of core/shell fibers. The processing conditions used for core/shell process add several requirements to classical electrospinning process itself. The key requirement is that shell polymer is based on electrospinnable solutions—the molecular weight, concentration, and entanglement of polymeric chains must be sufficient to produce stable fiber jets. On the other hand, the core solution could be a non-spinnable liquid (i.e., polymeric solution with low concentration). To produce core-shell fibers, the interfacial ten- sions between both liquid phases must enable drawing of core liquid and mixing of the two polymeric solutions must be avoided [53,60]. Coaxial electrospinning enables the development of composite functionalized nanofibers with a separated core/shell structure [53]. The main advantages of coaxial electrospinning include the possibility to form core-shell nanofibers from miscible and immiscible polymers, the high loading capacity of bioactive molecules, sustained release from the fibers, and a less harsh process that enables susceptible compounds to be delivered. The key advantage of coaxial electrospinning is the possibility to electrospun fibrous meshes from non-spinnable of hardly spinnable materials. The typical approach is using well electrospinnable polymer in the shell, which enables the formation of fibers from less electrospinnable solutions in core. For instance, zein nanofibers loaded with ketoprofen were prepared by coaxial electrospinning with polyvinyl pyrrolidone as a well-spinnable shell polymer [61]. Alternatively, zein nanofibers used for delivery of ibuprofen were prepared by modified coaxial electrospinning process [62]. The shell fluid was made of unspinnable dimethylformamide (DMF) solution, while core was made from spinnable zein ibuprofen solution. Because the zein ibuprofen solution showed problems with clogging upon blend electrospinning, employing of coaxial electrospinning enables overcoming of these problems and production of drug-releasing fine fibrous scaffold. 330 Core-Shell Nanostructures for Drug Delivery and Theranostics

Coaxial electrospinning was successfully used for delivery of broad spectrum of drugs. Core-shell fibers were prepared for delivery of antiinflammatory and analge- sic drugs (i.e., ketoprofen [61,63,64], acetaminophen [65], ibuprofen [62,66,67]), antimicrobial drugs (i.e., metronidazole [68,69], amoxicillin [70], curcumin [71], ampicillin [72], ciprofloxacin [73], tetracycline [74,75], vancomycin [76]), and anticancer drugs (i.e., cis-platinum [77], doxorubicin [78], paclitaxel [79,80], and 5- fluorouracyl [79,81]). Core/shell electrospinning is used for these molecules mainly because of prolongation of their release times. The coaxial electrospinning was showed to enable prolonged release and reduced burst compared to blend electrospinning [76,82]. Coaxial electrospinning enables slower release of small molecules from hydrophilic matrices. Yu et al. [82] prepared core-shell and monolithic nanofibers from zein loaded with ketoprofen. The release profile from non-coaxial fibers exhibited dominant burst release followed by negligible release of active molecule. On the other hand, core-shell fibers exhibited zero-order release of active molecules and prolongation of efficient times of delivery. In a similar study, fibers from PLGA, loaded with vancomycin, were prepared either by blend or coaxial electrospinning. In case of blend electrospinning, the release was faster at all time-points. The system was designed to release drug within 24 hours. However, core-shell electrospinning enables the formation of systems with long-term release properties. Jalvandi et al. [50] prepared levofloxacin-loaded PCL core-shell nanofibers for antimicrobial dressings. The system showed sustained release for 14 days with half- time of release about 3 days. The drug release rate is highly dependent on drug type, degradation of core-shell materials, and diffusion coefficients between core and shell layers. For instance, anticancer drug-loaded PCL/PVA core-shell fibers formed a structure with ultrathin core-shell fibers. However, the ultrastructure of fibers was based on the formation of continuous core combined with crystal clusters penetrating the shell. Upon release, the clusters turned into pores and facilitated faster release of drug [79]. In addition, coaxial electrospinning could be combined with other functionalization techniques [83]. Nanofibers prepared by coaxial spinning with tetracycline in core were further functionalized by covalent binding of proteins at the fiber surface. Such multifunctional system could be utilized for complex stimulation of tissue repair. The fast release of molecules from core could protect infection in implantation site, followed by long-term stimulation of proteins on the surface of fibers. From tissue engineering point of view, the key active molecules for stimulation of tissue repair and changing of cell phenotype are based on biopolymers. Biopolymers, such as DNA, RNA, and proteins show high susceptibility to unfavor- able environmental stimuli. Coaxial electrospinning is a gentle process for the encapsulation and protection of such structures. Core-shell nanofibers were shown to successfully deliver growth factors for tissue engineering [84 94]. PLGA nanofibrous membranes were used for co-delivery of dexamethasone and vascular endothelial growth factor (VEGF). The system enabled sustained release of VEGF for 28 days and stimulation of endothelial cells [90]. Similarly, PLCL was used for co- delivery of VEGF and platelet-derived growth factor (PDGF) for 28 days and enabled use as vascular implant in vivo [84]. Silk fibroin blend with PLA was used Blend electrospinning, coaxial electrospinning, and emulsion electrospinning techniques 331 for delivery of NGF and showed potential for nerve cell proliferation and differentiation to desired subtypes [94]. Similarly, aligned core-shell nanofibers with NGF were shown to help nerve regeneration in vivo [95]. A recent study [96] employed delivery of both NGF and monosialoganglioside 1 to stimulate Schwann cells proliferation. The co-delivery system showed improvement over delivery of solely NGF. PLCL was used for co-delivery of BMP-2 and dexamethasone. The work demonstrated prolongation of drug release upon encapsulation into the core. If the drug was encapsulated into the shell, the release was faster and accompanied with high burst during first hours [93]. Similarly, Yin et al. [97] prepared a co-delivery system for BMP-2 with IGF-I and showed osteoinductive and osteoinductive properties. bFGF was delivered by coaxial PLCL nanofibers [88]. The core of coaxial nanofibers was made of hydrogel or emulsion of PLGA with heparin. The system with emulsion core exhibited prolonged release in the first days after encapsulation. Core-shell fibers with hydrogel core, on the other hand, showed faster release due to increased diffusion through the core solution. In other work, PCL/ polyethylene oxide (PEO) coaxial fibers were used for delivery of bFGF. The sustained release was observed for 9 days, and enhanced fibroblast proliferation, collagen I production, and viability were observed on bFGF-releasing fibers [86]. To prepare a successful system for delivery of proteins, the preservation of the biological activity of the encapsulated molecules is particularly important. Coaxial electrospinning process enables the development of formulations avoiding harsh environment during encapsulation. Mickova et al. [59] developed a system for encapsulation of intact liposomes into core/shell nanofibers. Due to interfacial tension and short spinning times, the coaxial electrospinning enabled protection of liposomes from rupture due to high shear stress and the presence of nonpolar solvents in shell polymer (chloroform). Nevertheless, the results showed that despite improved bioactivity of model enzyme in coaxial fibers compared to blend fibers, the protein structure could be even more preserved in embedded intact liposomes. This results in improved retention of enzymatic activity in liposomal systems and highlights the importance of encapsulation conditions for susceptible biomolecules. Similar systems were developed using liposomes [98,99], alpha granules [99], or cells [100,101]. Coaxial electrospinning process was also used to perform fiber modification. Multiaxial electrospinning enables the formation of even more complex fiber morphologies. In triaxial electrospinning, the spinneret is composed of three separated needles [102]. However, setting of chemical and physical conditions is even more problematic in this methods [102 104]. Triaxial electrospinning was successfully used for delivery of doxycycline [102], nisin [105], and calcium phosphate nanoparticles [106]. This technology allows multiphasic delivery of active molecules with complex release profiles [107,108]. Moreover, the technology is useful for in situ synthesis of nanoparticles, i.e., by coprecipitation for calcium phosphate nanoparticles [106]. However, coaxial electrospinning process has several disadvantages. The production rate is rather limited. The process often results in fibers with different properties across the mesh layer. Finally, the compatibility of core and shell fluids must 332 Core-Shell Nanostructures for Drug Delivery and Theranostics be optimized for different active molecules, what leads to possible formation of artifacts and defects.

11.5.1 Emulsion electrospinning Numerous disadvantages of blend and coaxial electrospinning could be overcome by a novel approach called “emulsion electrospinning.” The technology combines both methods with emulsification approach. Emulsion electrospinning is based on single nozzle formation of core-shell structure. The core-shell organization of fibers is not introduced by the structure of the employed spinneret as in coaxial spinning but by electrospinning of stable emulsion of two or more fluids. Unlike in blend electrospinning, the emulsion is based on two or multiple phases, which are not mixed during the electrospinning process. The phases are immiscible and typically stabilized by proper surfactants. The electrospun solution is therefore based on different liquid phases—the continuous phase is converted into shell of fibers and the droplet phase is forming the fiber core. Processing conditions for emulsion electrospinning are partially different with respect to both blend and coaxial electrospinning. Generally, two types of emulsions are used for electrospinning process. Water- in-oil emulsions are based on lipophilic continuous phase and hydrophilic droplet phase [89,109]. This type of emulsion is used for encapsulation of polar and hydrophilic molecules, such as proteins. The emulsions are stabilized by surfactants with low hydrophilic lipophilic balance (i.e., Span 80, Span 60, Pluronics) [110]. The typical polymers for continuous phase are polyesters (i.e., PCL, PLA, and PLGA), polyurethanes (PUs), polystyrene, and other polymers soluble in lipophilic solvents. The core polymers are based on water-soluble polymers, such as PVA, PEO, polyvinylpyrrolidone, cellulose derivatives, chitosan, and alginates. The second type is oil-in-water (o/w) emulsions [111,112]. The continuous phase is formed by hydrophilic solution and droplet phase by lipophilic solution. The emulsion is stabilized by surfactants with high hydrophilic-lipophilic balance (HLB) value, such as Tween 20, Tween 80, and stearyl alcohol [110]. Beside these two systems, multiple emulsions, such as water-in-oil-in-water (w/o/w), are produced by additional emulsification of o/w emulsion in water phase. Such multiple emulsions have higher requirements such as proper surfactant selection and emulsification protocol optimization. Optimization protocols include multiple variables and should follow quality- by-design strategies described by Badawi et al. [113]. Besides emulsion formation, other properties are crucial to achieve successful electrospinning process. The emulsion must be formed by electrospinnable continuous phase polymer. This means that the solution must have sufficient conductivity, a suitable polymer concentration, and the polymer must have an enough high molecular weight and surface tension. On the other hand, the properties of droplet phase are affecting internal organization of fibers. Core solution morphology is governed by its viscosity as result of polymer properties. If the viscosity of droplet phase is higher than the viscosity of continuous phase, the emulsion electrospinning process results in the formation of continuous core along the fiber axis [114]. On the other hand, if the core Blend electrospinning, coaxial electrospinning, and emulsion electrospinning techniques 333 has lower viscosity than shell phase, the core phase breaks into separated droplet and the core has noncontinuous morphology [115]. The phenomenon is connected with the reduction of polymer chain entanglement and cohesion in less viscous solution as a result of lower polymer concentration, molecular weight of chain bonding. Therefore, upon elongation of fibers from polymeric solution, the core droplets tend to break up into separated smaller droplets. Similarly, surfactant type was shown to affect the organization of core in emulsion electrospinning [116,117]. The core morphology is among the most crucial variables affecting the release kinetics from emulsion nanofibers. As in other nanofiber types, release from emulsion nanofibers is governed by diffusion or degradation mechanisms [118]. Degradation mechanism is dominant in fibers from degradable materials. Upon degradation, the drugs are released from polymeric structure depending on dissolution of polymeric matrix [2,119]. The drug release from materials, which are nondegrad- able in the requested time frame, is governed by diffusion rate through polymeric matrix [120]. In case of emulsion electrospinning, typically a combination of both mechanisms takes place. In case of typical W/O emulsions, the shell of fibers is made of polymers stable in water (i.e., PLGA), while the core is made from water- soluble polymers (i.e., PVA). The rate limiting factor is therefore the diffusion rate of water and drugs through polymer shell. The rate depends on the internal structure of fibers and on the number of contact points available for the solvent to dissolve the core polymer. In case of continuous core, the release is governed by capillary forces inside core-shell fibers and flux of drug governing diffusion gradients in fibers. In case of fibers with noncontinuous cores, the release depends on the inter- connection of droplets [121]. The solvent first dissolves the droplets on the surface of fibers resulting in rapid release of their cargo. If the droplets are not intercon- nected or the solvent diffusion is slowed down, the availability of drug decreases. In case of droplet interconnectivity, the solvent could reach droplets deeper in fibers and result in sustained release of active molecules. Nevertheless, the stability of the emulsion is important for fiber internal morphology and release properties; if the stability of the emulsion is low, the core droplets tend to agglomerate, accumu- late on the surface of fibers, and produce a nonhomogenous release pattern with a huge initial burst [114]. One of the strategies to overcome rapid release of active molecules is the utilization of week electrostatic forces. Because most of the active molecules (i.e., proteins) bear charge, incorporation of species with opposite charge inside emulsion fibers results in increased interaction and slower release from fibers. For instance, cellulose acetate was used for prolonging release of growth factors from PLGA fibers [89]. From the drug delivery point of view, the emulsion electrospinning was utilized for the delivery of numerous active molecules. Combination of coaxial and emulsion electrospinning was utilized for development of levetiracetam-releasing fibers [122]. The system, enabling sustained released over 20 days, has the potential for the development of novel more convenient epilepsy therapy. Small pro-osteogenic molecules, such as acorbate-2-phospate were encapsulated into emulsion spun fibers [123]. The emulsion nanofibers showed more sustained release profile compared to coaxial nanofibers. However, the release rate from emulsion fibers was faster than 334 Core-Shell Nanostructures for Drug Delivery and Theranostics from blend spun fibers indicating improved availability of drug due to clustering of active molecule to core droplets. Oil-in-water emulsions based on PVA were used for encapsulation of fragrances. For instance, limonene was embedded into PVA fibers by emulsion electrospinning. The technology enabled release of fragrance for 15 days [124]. Emulsion electrospinning was used also for delivery of antibiotics (i.e., vancomycin [125]), anticancer drugs (i.e., doxorubicin [114,126], hydroxycamptothecin [127], and paclitaxel [128]), and antiinflammatory drugs (i.e., cele- coxib [129] and ketoprofen [130]). The technology is especially useful for delivery of susceptible molecules [131 133]. Emulsion electrospinning could maintain the activity of delivered proteins, as was shown on the model of horseradish peroxidase [134], lactase [135], and lysozyme [136]. Emulsion electrospinning was utilized for delivery of NGF- stimulating neural cells [109,137]. Similarly, emulsion electrospinning was used for the development of vascular grafts based on the release of growth factors [138 141] or peptides [142]. PLGA scaffolds prepared by negative voltage emulsion electrospinning were used to fabricate long-time releasing system for VEGF [143]. Classical emulsion electrospinning was able to produce systems releasing VEGF for 28 days [139,140]. System co-delivering VEGF and heparin were made by emulsion electrospinning with PLCL with potential use in cardiac tissue engineering [144]. Similarly, a system co-delivering VEGF and PDGF was prepared by emulsion electrospinning of PLCL [138]. PLGA fibers with cellulose acetate were used for the delivery of bFGF. The amount of cellulose acetate regulated release of bFGF by altering the electrostatic interaction between active molecule and fibers [89]. Emulsion electrospun PCL scaffold with bFGF was showed to enable sustained delivery of bFGF, stimulate cell proliferation in vitro, and improve bone regeneration in vivo [145]. Osteoinductive fibers were also prepared by emulsion electrospinning of PLGA nanofibers-releasing collagen [146] and collagen-derived peptide [147] Emulsion electrospinning of fibers-releasing PDGF were used for the stimulation of mesenchymal stem cell osteogenic differentiation. The loading of growth factor was increased with higher concentration of droplet polymer concentration [148]. Dual- source spinning enabled the formation of composite fibers with core phase based on either emulsion encapsulated BMP-2 or dispersion of calcium phosphate [149]. Bicomponent systems have the potential for use in bone tissue engineering, as was demonstrated in an in vitro study [150]. PU fibers loaded with PDGF were used for stimulation of tenocyte proliferation in vitro [151]. BFGF-releasing emulsion fibers were used as a scaffold for rotator cuff repair [152] and tendon repair [153]. Similarly, emulsion fibers were used for the formation of EGF releasing scaffolds for skin tissue engineering [154,155]. Emulsion spun PCL scaffold loaded with a combination of hyaluronanu, and EGF was shown to improve would healing in vitro and in vivo [156]. Similarly, system based on bFGF encapsulation was showed to stimulate wound healing on diabetic model [157]. Dispersion electrospinning also enables micro and nanoparticles to be loaded into polymeric nanofibers. Antibacterial properties were achieved by dispersion electrospinning of nanoparticles with vancomycin [125]. Similarly, magnetic Blend electrospinning, coaxial electrospinning, and emulsion electrospinning techniques 335 nanoparticle-loaded fibers were prepared by dispersion electrospinning [158]. Dispersion electrospinning was used for delivery of bFGF in systems combining chitosan nanoparticles with growth factor, PVA, and chitosan electrospinning [159]. Moreover, hydroxyapatite as an osteoinductive material was combined with nanofibers using dispersion electrospinning [160]. Hydroxyapatite particles were able to preserve the activity of BMP-2 loaded in electrospun PLGA scaffolds, as proteins can attach to these hydrophilic particles [59]. These results show that emulsion electrospinning is an appropriate way to produce core-shell nanofibers loaded with various bioactive molecules. Nevertheless, emulsion electrospinning also suffers from one main disadvantage: it does not work properly for polymer solutions with small interfacial tension. In addition, the preparation of emulsions by mechanical mixing or ultrasonication has to be carried out with care, as this process can affect the structure of embedded molecules or polymers [60,61].

11.6 High-throughput electrospinning technologies

The advanced drug-loaded nanofibers were shown to deliver a wide range of active molecules. However, the technologies are facing numerous challenges for a wide application of systems in everyday life. Electrospinning is a fiber-forming process, which enables the formation of fibrous mesh. However, the throughput of classical needle electrospinning protocols is rather limited and reaches only about 0.001 0.1 g/hour [10,161]. This limitation is due to the need for optimal polymer solution flow rate, which is in the order of 10 100 μL/minute for classical needles. Recently, methods for improving electrospinning throughput were developed. The simplest possibility is the multiplication of needles to increase the number of fiber jets and throughput of process [162 167]. The needle arrangement plays role in the organization of fiber jets and resulting fiber properties, such as uniformity and diameter. The systems with linear, triangular, and square needle organization were developed to produce uniform nanofibers [163]. Similarly, system with hexagonal needle arrangement enables improved fiber jet distribution and spinning quality. In addition, shielding ring—a metallic ring around electrodes—was used to decrease fiber collecting area and improved homogeneity of electrospun fibers [168]. The key disadvantage of multineedle electrospinning is the problem with finding of optimal needle arrangement, applied voltage, and collector/electrode distance [162 167,169,170], because multiple needles are forming nonhomogenous electric field resulting in change of fiber jet properties. The way to change field arrangement is variable among different electrospinning solutions. For instance, the optimal voltage and needle arrangement varies depending on polymer type, solution concentration, surface tension, and conductivity. To find optimal needle arrangement, finite element analysis was used to model multineedle electrospinning process [169 171]. 336 Core-Shell Nanostructures for Drug Delivery and Theranostics

Due to problematic optimization of multineedle methods, needleless electrospinning methods overcoming the problems of optimal needle arrangement were developed. The theory of needleless electrospinning is based on self-arrangement of fiber jets on surface of liquid due to static wave of surface tension and electrostatic forces [8]. Needleless electrospinning could be performed on a range of electrodes. Rod electrodes were based on spinning from polymeric drop with higher diameter enabling the production of multiple fiber jets [8,172]. The spacing of fiber jets is changing with the variation of electrospinning variables and enables optimal jet spacing. The second type of needleless electrodes is based on the rotation of drum, disc, or coil electrodes in polymer bath [173]. Upon rotation, the polymer forms thin layer and on the top of rotating element forms multiple fiber jets. The initial technology is based on the work of Jirsak et al. [174] and commercialized under trade name Nanospider. The modification of technology is based on electrospinning from rotary disks and coils [175 181]. Disc and coil electrodes were shown to improve the quality of nanofibrous scaffold compared to needle electrospinning methods [182,183]. Wang et al. [184] evaluated electric field distribution on various needleless electrodes and found increased electric-field intensity at disc electrodes compared to cylinder electrodes. Therefore, high-throughput and improved electrospinning properties could be achieved by such systems. The addition of rods con- centrating electric field leads to increase of electrospinning throughput [185]. The key disadvantage of systems with rotary electrodes is evaporation of polymer from liquid bath and change of properties during electrospinning process. Alternatively, static electrodes with improved polymer filling were developed recently. Pejchar et al. [186] developed a range of linear slit electrodes. The electrodes are filled by polymer from bottom port and enable the formation of polymeric layer on the ori- fice of slit between two metallic plates. Static needleless electrodes could be also based on pyramid spinneret [187]. The polymer solution cascades through steps of pyramid and on edges forms multiple fiber jets. Static electrodes enable continuous production of nanofibers with higher throughput and facilitate industrial application possibilities. The throughput of electrospinning technology could be increased by electroblowing [188]. Electroblowing is based on the application of air stream on the electrode leading to simplified drawing of fibers. The airstream enables improvement of electrospinning throughput, fiber homogeneity, and decreased morphology [189]. High-throughput applications of core-shell nanofibers were also reported recently. Forward et al. [190]. developed a needleless coaxial electrospinning method based on the formation of a bilayer of two immiscible liquids on the surface of a rotation wire electrode. The electrode is first immersed in a core polymer followed by immersion in a shell polymer. The bilayer stability is based on liquid immiscibility, density, and dielectric constants. Upon the action of electrostatic forces, a composite coaxial jet emerges on the surface of the fibers. On the other hand, Vyslouzilova et al. [60] introduced a system based on dual-slit electrodes. Two slits, coaxially arranged, enabled the formation of liquid bilayer from both immiscible and miscible polymers. The resulting coaxial nanofibers had improved drug release properties and higher core loading compared to classical coaxial Blend electrospinning, coaxial electrospinning, and emulsion electrospinning techniques 337 nanofibers. Needleless electrospinning was also used for the production of fibers from emulsions. Emulsion electrospinning from a needleless dual-wire electrode was used for high-throughput production of core/shell fibers [191]. Needleless static wire electrode was used for the formation of core/shell by electrospinning of PCL/ Pluronic F68 emulsions [134]. The produced nanofibers were loaded with model enzymes, and the technology showed improved preservation of enzymatic activity, scalable release times, and improved production rate. Scaffolds loaded with growth factor enable large scale production of systems for mesenchymal stem cell proliferation. Alternatively, core-shell nanofibers could be produced at high throughput by recently developed emulsion centrifugal spinning [121]. The technology is based on the formation of ultrathin fibers by the application of high centrifugal forces on polymeric solution. The use of stabilized emulsions enabled the formation of core- shell nanofibers. The fiber mesh had more 3D morphology with opened pores and supported protein bioactivity protection, regulated release, and stimulation of osteo- blast proliferation. Altogether, the high-throughput production methods for core-shell nanofibers are increasing the potential for practical use of developed scaffolds. The technologies will make the drug-loaded fibrous scaffold more cost efficient and available in industrial quantities. Core-shell nanofibers were studied as advanced drug delivery and scaffolding system in various laboratory settings and have enormous potential to solve numerous health problems. However, additional considerations on the use of medical grade polymers, excipients, and drugs are necessary for rapid translation from research to practical use.Acknowledgement: This project was supported by the Czech Science Foundation Grant No.18-09306S. This project has recieved funding from the MSCA RISE program under grant aggreement No. 691061 (NanoBAT) and No. 778098 (NanoFEED).

Acknowledgment

This project was supported by the Czech Science Foundation Grant No.18-09306S. This project has recieved funding from the MSCA RISE program under grant aggreement No. 691061 (NanoBAT) and No. 778098 (NanoFEED).

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