Electrospun Nanofibers for Antibiotic Release and Antibacterial Performance: A Nanomanufacturing Perspective

Abstract

Electrospun composites are desirable materials for drug delivery applications. Regarding microbial infections as a case study, the antibacterial effect is enhanced by physical attributes of electrospun meshes, namely, a high surface area-to-volume ratio and porosity, 3D topography, and customized surface functions. Beyond mimicking nanostructured fibers, the delivery of antibiotics from such composites enhances antibacterial efficacy, sustained release kinetics, and reduced wound infection while minimizing side effects. Concern over antibiotic resistance and the insufficient availability of pharmaceutical agents for effective infection treatment is increasing worldwide. A significant number of publications have reported the fabrication of electrospun composites to mitigate bacterial pathogenesis. However, from a structural and morphological perspective, the implications of electrospinning approaches for antibiotic delivery have not been reviewed. This proposal presents a comparative study of the different assemblies induced by electrospinning, enabling the development of platforms for administering antibacterial agents. The primary objective is to conduct a comprehensive examination of the considerations involved in electrospinning-based manufacturing of drug delivery systems and antibiotic loading, ensuring a thorough design process that accounts for composite processability, monitoring methods for kinetic behavior analysis and modeling, and biological considerations for pre-clinical in vitro characterization.

1. Introduction

The terms antimicrobial, antibacterial, and antibiotic are often used interchangeably in the scientific literature, even though they represent distinct pharmacological and functional concepts, which can lead to conceptual ambiguity and misinterpretation of experimental outcomes. Antibacterial agents are compounds that target bacterial organisms, whether of natural or synthetic origin, and are often characterized by their bacteriostatic or bactericidal properties [1]. In contrast, the term antibiotic traditionally denotes compounds produced by microorganisms or derived from them that exert activity against bacteria. However, its use has broadened clinically to include related synthetic or semi-synthetic agents directed against bacterial infections [2]. In this review, we adopt and apply these definitions—antimicrobial as the overarching category, antibacterial as bacteria-specific agents, and antibiotic as the subclass of naturally derived or related agents—to ensure clarity and scientific precision throughout the manuscript.

Antibiotics are among the most prescribed drugs in human and animal medicine, serving as powerful tools for treating bacterial infections; however, they are also misused to treat viral diseases. Repeated exposure to these drugs, especially at low doses, contributes to the spread of antibiotic-resistant bacteria, making infections more challenging to treat. Organic antibacterial agents can be classified by nature (natural or synthetic), as shown in Figure 1.

Antibiotic resistance has necessitated the development of new and improved antibacterial agents. Improving the precision of existing antibiotic therapies by optimizing dosing and therapy duration can reduce the risk of toxicity and minimize the development of drug resistance. Low bioavailability, side effects, poor penetration, and accumulation at the site of infection are other factors that contribute to antibiotic resistance and the clinical failure of antibiotic therapy [3]. In addition, the development of new antibiotics could be based on the chemical structures of existing ones; however, their mechanisms of action are the same as those to which bacteria have already developed resistance. Therefore, approaches to the stewardship of existing antibiotics are needed [3].

In this context, the use of plant-derived antibacterial compounds (such as alkaloids, coumarins, essential oils, flavonoids, lectins, phenolics, polypeptides, terpenoids, and tannins) [4] is a promising alternative for controlling bacterial infections and mitigating the emergence of antibiotic resistance, since natural products have high potential against resistant infections due to their multimodal mechanism of action [5].

Investigations related to the development of new and efficient antibiotic delivery systems include siderophores, antibodies, bacteriophages, short cell-penetrating peptides, host innate antimicrobial peptides, and nanotechnology-based delivery systems [3]. Nanotechnology-based delivery systems could improve treatment for drug-resistant bacteria and reduce side effects by using nanocarriers such as liposomes, polymer nanoparticles, polymer nanofibers, and nanogels. These are some examples of targeted delivery designs based on nanotechnology, improving solubility and controlled release, enhancing bioavailability, bearing the entry into bacterial cells and biofilms, and thereby improving drug efficacy [6].

Nanogels are composed of soft hydrogel nanoparticles that can encapsulate and deliver antibiotics more effectively while protecting them from degradation [7]. Antibiotics released from nanogels maintain therapeutic levels over time and reduce the frequency of dosing [8]. Drug penetration and efficacy are enhanced by nanogels’ high-water content, biocompatibility, tunable particle size, and stimulus-responsive properties. Scalability, reproducibility, biosafety, immunogenicity, and stability in complex biological environments are concerns that hinder clinical translation [7,9,10].

Polymer nanoparticles loaded with antibiotics can increase drug concentration and reduce systemic side effects. They can be engineered to target specific pathogens or infected cells, offering potential advantages in treating bacterial infections, especially those caused by multidrug-resistant bacteria. Biocompatible and biodegradable polymer nanoparticles protect drugs from degradation, can improve their solubility, enhance their penetration into target tissues, and facilitate controlled release at the site of infection [11,12]. They can be delivered via different routes, including lung drug-delivery; however, biodegradability and nanoparticles’ safety are concerns on this delivery route [12]. Some limitations of these delivery systems are related to particle stability and aggregation, premature drug release before reaching the target, and potential toxicity of the polymers and the used solvents, in addition to difficulties in scalability, regulatory compliance, and controlling drug release kinetics to prevent leakage or ensure sustained delivery [13,14,15].

On the other hand, electrospun polymeric nanofibers loaded with antibiotics can fight against bacteria by effectively delivering drug cargo to the target site. Topographic and unique physicochemical properties, including bacteria-like nano-scale features, are key factors in enhancing bacterial attachment and inhibition. A large surface area-to-volume ratio facilitates the encapsulation of antimicrobial therapeutic agents and allows high interconnected porosity, which enables higher loading of antimicrobial agents, increases bacterial attachment to their surfaces, promotes oxygen and nutrient exchange, ensures sustained drug release, and enhances wound healing by promoting cell proliferation. Surface chemistry also influences their therapeutic activity and offers tailored antimicrobial therapy. Their nanometric-scale structure resembles the native extracellular matrix (ECM) architecture and provides an excellent environment for cell migration, adhesion, differentiation, and regeneration [16]. Nanofibers have a remarkable drug-loading efficiency and adaptability in enabling efficient drug administration.

Ultrafine polymer nanofibers are commonly produced by melt blowing [17], phase separation [18], centrifugal spinning [1], template synthesis [19], and electrospinning [20]. Among them, electrospinning is the simplest and most effective method for their manufacture. Due to the versatility of the electrospinning setup, the morphological features of polymer nanofibers can be tailored by tuning polymer solution parameters or processing parameters. The final characteristics related to size, diameter, porosity, surface wettability, and surface charge of nanofibers are crucial to induce a bactericidal effect [16].

Due to the high potential of nanofibers as drug-delivery systems for antimicrobial agents, this review compares various electrospun assemblies used to fabricate platforms for the delivery of natural and synthetic antibacterial agents. Beyond electrospinning implications, a perspective for kinetic analysis and in vitro procedures is also outlined.

2. Nanofiber Manufacturing

Electrospinning is a voltage-driven process for producing micro- and nanofiber collections from polymeric solutions. The typical electrospinning setup (shown in Figure 2) includes four main components: a high-voltage power supply, a syringe pump, a spinneret, and a collector. The polymer solution is charged in a syringe and pumped at a determined flow rate. An electrostatic force is induced between the needle tip and the target ground electrode (the collector) with a sufficient voltage to overcome the solution surface tension, forming the Taylor cone and elongating the jet, which travels from the needle to the collector, reducing its diameter and increasing its length. The use of a volatile solvent in the polymer solution is significant for this process, as it enables solvent evaporation. In contrast, the polymer solidifies and deposits as fibers on the collector.

The control of nanofiber morphology and diameter relies mainly on process parameters, such as voltage, distance between the needle and the collector, solution flow rate, collector type, and ambient conditions, which affect fiber diameter and bead formation. Voltage is crucial for drawing the polymer solution into a jet and subsequently forming nanofibers [21,22]. Precise control of the polymer solution flow rate ensures a consistent, controllable electrospinning process [23]. The spinneret, which is typically a metallic needle, acts as a nozzle through which the polymer is ejected [22,24], and the collector can influence orientation and morphology [24,25]. Other solution parameters, such as concentration, viscosity, molecular weight, surface tension, conductivity, and solvent type, can, in addition to affecting fiber diameter and the production of good-quality nanofibers without beads, lead to no fiber formation [20].

Electrospinning is recognized as a versatile technique for creating random or aligned nanofiber mats from diverse materials, with controlled microstructures and morphologies, including solid, porous, or hollow structures. Single-phase, embedded with nanoparticles or active compounds, surface-porous, multiaxial, or hollow nanofibers can be produced. This diversity is achieved by modifying the polymer solution or the electrospinning setup. Nanofiber mats possess a high surface area-to-volume ratio and a high degree of interconnected porosity. Control of fiber diameter and alignment [26] enables potential functionalization [26,27], compositional versatility, and customizable properties by adjusting the material system and processing parameters [28,29]. Thus, their unique microstructure makes them excellent candidates for use as drug carriers in drug delivery systems (DDS).

DDS and drug carriers are crucial for enhancing drug efficacy and safety. Key functions of DDS include controlled release, target delivery, enhanced bioavailability, drug protection, and overcoming biological barriers.

2.1. Drug Delivery Systems (DDS) Based on Nanofibers

DDS are formulations or technologies for the safe and effective transportation of active pharmaceutical ingredients (API) or drugs into the body to enhance the desired therapeutic effect [30]. Key functions of DDS include controlled release, target delivery, enhanced bioavailability, drug protection, and overcoming biological barriers [31]. These systems can influence a drug’s pharmacological activity by modulating its release from the carrier. In addition, incorporating a drug into a DDS can reduce the frequency of administration and toxicity [32].

DDS based on nanofiber mats can encapsulate and release drugs in a controlled manner; furthermore, they can be designed in different forms for administration via topical, oral, and transdermal routes [32,33,34,35]. The drug-loading technique depends on the drug solubility in the polymer solution and is crucial to the release process. Drugs can be encapsulated into nanofibers using a single-phase electrospinning process, chemically immobilized on fiber surfaces, or loaded by physical adsorption via electrostatic, hydrophobic, hydrogen-bonding, van der Waals forces, and fiber-surface interactions [36].

Incorporating antibiotics into DDS could improve bioavailability and target efficiency to address antimicrobial resistance, focusing on improved treatment strategies [37]. The nanofiber structure promotes surface interactions and enhances bacterial inhibition [38]. Antibiotic drugs are commonly incorporated into polymer nanofibers through simple immersion of the nanofiber membrane into the corresponding antibiotic solution [39], by mixing components into the solution before electrospinning [40,41,42] or by covalent bond to the nanofiber surface [43]. The resulting antibiotic-loaded nanofibers can also be used as coatings for metallic implants to prevent and treat bone infections [44].

2.2. Electrospun Polymers for Antibiotic Loading

Electrospinning’s versatility also stems from the ability to produce antimicrobial electrospun nanofibers from natural or synthetic polymers. Standard polymeric matrices for antibiotic drug delivery include natural polymers (such as polysaccharides and proteins), synthetic polymers, and, to improve properties, natural/synthetic mixtures.

2.2.1. Polysaccharides

Chitosan (CS) [45] is an antibacterial, biocompatible, non-toxic, and biodegradable biopolymer [46]. Pristine chitosan nanofibers showed promise for wound dressings; however, they have limitations in broad-spectrum antibacterial activity and mechanical properties [47]. It is a poorly electrospinnable polymer due to increased repulsive forces between polymer ion groups during electrospinning, which leads to the formation of bead-like particles [38]; therefore, it is commonly blended with a secondary electrospinnable biocompatible synthetic polymer, such as polyvinyl alcohol (PVA) [48], polyvinylpyrrolidone (PVP), or polyethylene oxide (PEO) [49] to improve the spinnability and mechanical properties of nanofiber mats. By electrospinning, chitosan-based nanofibers can be produced using binary solvent systems of acetic acid/water [50], acetic acid/1,1,1,3,3,3-hexafluoroisopropanol (HFIP) [51], and trifluoroacetic acid/dichloromethane (DCM) [52], resulting in a fiber diameter of 200–400 nm [50,51].

Cellulose and its derivatives are another family of electrospinnable polysaccharides. Cellulose is not intrinsically antimicrobial, so it must be chemically modified to turn it into an antimicrobial agent, e.g., by increasing the alkyl chain length of the aminoalkyl groups or the number of amino groups. Chemical modifications are performed on the surface of cellulose fibrils by reacting through the hydroxyl groups of cellulose with carboxylic acids, amines, or aldehydes [53]. Cellulose and its derivatives can be electrospun from their corresponding solutions in binary solvent systems of dimethyl carbonate (DMC) and cyclopentanone (CPO) [54], dimethylacetamide (DMAc) and dimethylformamide (DMF) [55], and superbase-derived ionic liquid (SIL) [56] to produce non-woven mats of diameters of 100–500 nm [57], depending on the composite components.

Hyaluronic acid (HA) is another polysaccharide that has been loaded with antimicrobial agents due to its biocompatibility and biodegradability. In addition, HA has been shown to possess intrinsic antimicrobial properties, thereby increasing its biomedical value [58]. It is implicated in physiological functions, including maintaining the extracellular space to regulate osmotic pressure, lubrication, and promoting cell repair [59]. HA is commonly electrospun using distilled water as a solvent, rendering fiber diameters of 100–300 nm [60], and encapsulation capacity of bioactive molecules ranging from 70 to >90% [60] as typical attributes.

To a minor extent, sodium alginate has been incorporated into nanofibers loaded with essencial oils, such as those from oregano [61] cinnamon, clove, and lavender [62]. This polysaccharide is useful due to its hydrophilic nature, which provides rapid tissue granulation and re-epithelialization under moist environments [63]. The fabrications of sodium alginate nanofibers often implies blending with synthetic and natural polymers, such as PEO [64,65], PVA [66], and chitosan, to produce vancomycin [64], or levofloxacin-loaded systems [67]. Sodium alginate electrospinning from distilled water [64] has been reported to produce a fiber diameter of around 100–900 nm [65,66].

Pullulan is another natural polysaccharide produced by Aureobasidium pullulans in starch crops that can form electrospun nanofibers [67]. Pullulan-based nanofibers have been loaded with tetracycline [68], cinnamaldehyde [69], and different antimicrobial peptides [70,71,72], showing bactericidal activity against S. Typhimurium, L. monocytogenes, and L. mesenteroides [72]. From aqueous solutions, pullulan can be processed to obtain fibers with diameters of 200–600 nm [73,74].

2.2.2. Proteins

Zein [42], poly (L-lysine) [75,76], whey protein [77], and casein [78,79] are examples of proteins used to craft biocompatible nanofibers. However, due to their low spinnability, these materials are commonly blended with synthetic polymers to improve the mechanical properties of the resulting nanofibers.

Gelatin nanofiber electrospun dressings containing ε-polylysine crosslinked with polydopamine have been successfully tested for treating second-degree burns in a porcine model, promoting wound closure, reducing hypertrophic scarring, and decreasing bacterial bioburden in porcine models [80]. The co-electrospun zein and collagen nanofiber mat has been used as a dressing to cover full-thickness skin wounds in mice, promoting rapid tissue regeneration [81].

2.2.3. Synthetic Polymers

Synthetic nanofiber mats based on biocompatible polymers enriched with antibiotics have also been produced [82,83]. The most used synthetic polymers have been polycaprolactone (PCL), PVA, PEO, poly (D,L-lactic acid-co-glycolic acid) (PLGA), poly(L-lactide-co-D,L-lactide) (PDLLA), and polylactic acid (PLA); some of them have been applied to prevent bone infections and used as coating for implants [84].

In general, synthetic polymers have also been enriched with essential oils to develop antimicrobial agents [85,86], most of which are used for food packaging applications [87] or even for cancer treatment [86].

2.2.4. Natural/Synthetic Polymer Combinations

Nanofiber composites combining natural and synthetic polymers have been commonly produced and tested for bacterial inhibition, as is the case with the chitosan/PEO/bacterial cellulose nanofibers composite [88], which has shown reduced antibacterial activity against E. coli and S. aureus, ascribed to the high hydrophilicity and crystalline structure of bacterial cellulose. PCL/gelatin nanofiber mats loaded with metronidazole and used as subcutaneous implants in rabbits for 8 months have exhibited good biocompatibility, and increasing the gelatin proportion enhances cell adhesion and proliferation [89]. Polyurethane/cellulose acetate/zein composite mats have been investigated as wound dressings; to prevent common clinical infections, incorporating streptomycin sulfate into electrospun nanofibers that demonstrated antibacterial activity against either Gram-positive and Gram-negative bacteria [90].

Zein/PCL electrospun matrices loaded with tetracycline hydrochloride have efficiently inhibited the growth of S. aureus, displaying sustained antibiotic release to treat and prevent bacterial infections [79]. The sustained release of tetracycline from these matrices, in addition to efficient in vitro destruction of S. aureus, has also demonstrated antibacterial activity against ATCC 25923 in an ex vivo pig skin model [91]. Core–shell nanofibers composed of PCL in the shell surrounding the zein core containing tetracycline hydrochloride have shown cytocompatibility, enhanced fibroblast cell attachment, and antimicrobial activity against E. coli and S. aureus [92].

Using poly(L-lactide-co-ε-caprolactone) (PLCL) as the base biomaterial to produce electrospun nanofibers, which have been surface modified with tannic acid and polylysine to yield a dressing with exceptional cytocompatibility and a synergistic antibacterial-anti-inflammatory effect for infected wound healing [75].

Casein/PVA blends containing the antimicrobial compounds, octenidine and polyhexanide have been used to produce nanofiber mats for multifunctional antimicrobial wound dressings with potent activity against S. aureus, S. pyogenes, E. coli, and C. albicans [78]. While casein/PEO electrospun mats incorporating tannic acid inhibit the growth of S. aureus and E. coli [79] and PEO/casein nanofibers loaded with thymol and β-cyclodextrin inclusion complexes have presented long-term antimicrobial activity for chilled beef preservation [93].

Most antibiotic-loaded DDS based on random nanofiber mats have been studied for wound-dressing applications and implant-associated infections. They are based on polymer mixtures of natural and synthetic polymers and exhibit antibacterial activity against Gram-positive and Gram-negative bacteria (

Table 1. Antibiotics-loaded nanofibers for wound dressing applications and implant-associated infections.

Wound Dressing Applications
Polymer Matrix	Type of Microstructure	Antibiotic/ Antibacterial	Against	Ref.
SA/CS Core: PCL/collagen Shell: doxycycline/PEO	Three-layer/aligned	Doxycycline	Not reported	[94]
CS/SA	Random mats	Levofloxacin	S. aureus, P. earuginosa	[63]
CS/PEO	Random mats	Ciprofloxacin	S. aureus, E. coli	[95]
CS/PEO	Random mats	Teicoplanin	S.aureus	[49]
Gelatin	Random mats	ε-polylysine	P. aueruginosa and S. aureus	[80]
Zein/collagen	Random mats	Berberine	S. aureus and E. coli	[96]
Zein/PCL	Random mats	Tetracycline	S. aureus	[97]
Zein/PCL and Zein/PEO	Core–shell	Tetracycline	S. aureus and E. coli	[92]
Casein/PVA	Random mats	Octiset® and polyhexanide	S. aureus, S. pyogenes, E. coli and C. albicanis	[78]
PCL/gelatin	Random mats	Trimethoxysilylpropyl octadecyldimethyl ammonium chloride	S.aureus, P. earuginosa	[98]
Fish Scale Gelatin	Random mats	Helichrysum italicum and Lavandula latifolia	S. aureus, E. coli and C. albicanis	[99]
PDEGMA/P(LLA-CL)	Random mats	Ciprofloxacin	S. aureus, E. coli	[100]
SA/PVA/PEO	Random mats	Berberine	E. coli	[101]
PVA/HA/cellulose nanocrystals	Random mats	L-arginine	K. pneumonia	[102]
Nylon 6/HA/CS	Random mats	none	S. aureus, E. coli	[103]
CMC/PVA	Random mats	Colistin	S.aureus, E. coli, K. pneumonia, P. earuginosa	[104]
HA	Random mats	ε-polylysine Cinnamon essential oil	S. aureus, E. coli S. aureus	[59] [105]
PEO/HA	Random mats	Kanamycin	L. monocytogenes	[106]
PU/cellulose acetate/zein	Random mats	Streptomycin sulfate	S. typhimurium, E. coli, V. vulnificus, S. aureus, B. subtilis	[90]
PLCL	Random mats	Tannic acid and polylysine	S. aureus	[75]
Implant-associated infections
PLGA	Random mats	Fusidic acid/rifampicin	S. aureus, S epidermides, MRSA and MRSA (Newman)	[84]
PLGA	Random	Vancomycin	S. aureus	[107]
PGLA-loaded deproteinized bone	Random	Vancomycin	S. aureus	[108]
PCL	Random	Metronidazole	F. nucleatum	[109]
PDLLA	Random	Amoxicillin	S. sanguinis and P. gingivalis	[110]
PLGA/PCL	Random	Vancomycin/Rifampicin/Linezolid/Daptomycin	S. aureus	[111]
PLGA/PCL	Random	Linezolid	S. aureus	[112]

CMC: carboxymethyl cellulose, CS: chitosan, HA: hyaluronic acid, PVA: polyvinyl alcohol, PCL: polycaprolactone, PEO: polyethylene oxide, PU: polyurethane, PLCL: Poly(L-lactide-co-ε-caprolactone), PDEGMA: poly(di(ethylene glycol) methyl ether methacrylate), P(LLA-CL) and poly(l-lactic acid-co-ε-caprolactone), SA: sodium alginate, PLGA: D,L-lactic acid-co-glycolic acid.

).

Although antibacterial nanomaterials have multiple mechanisms of action (e.g., physical membrane damage, ROS generation, and ion release), scientific evidence indicates that bacteria have a high adaptive capacity. Recent studies suggest that repeated exposure to nanostructures can induce defensive mechanisms, such as efflux pumps, changes in membrane permeability, the production of protective biopolymers, and the formation of denser biofilms, thereby reducing their susceptibility. These adaptations do not always equate to classic resistance, but they do represent induced tolerance to prolonged selective pressure [113,114].

Furthermore, it has been documented that the mechanisms of adaptation to nanomaterials share similarities with antibiotic resistance, including the activation of the SOS response and the genetic regulation of stress proteins. Systematic reviews have shown that sublethal exposure to metal nanoparticles and metal oxides can select strains with greater tolerance, suggesting a real evolutionary risk if these technologies are used extensively and without control [114,115].

In the specific case of nanofibers, there are no clinical reports of bacterial resistance directly induced by these systems. However, because some nanofibers release active agents gradually, they can generate subinhibitory gradients like those of conventional antibiotics, favoring adaptive processes. Therefore, although no widespread clinical phenomenon is currently observed, the literature warns that chronic selective pressure could induce long-term microbial tolerance [113,116].

3. Antibiotic Loading Mechanisms

In general, polymeric electrospun nanofibers are excellent platforms for drug delivery purposes [36,44,117,118,119,120]. The most popular strategies for loading drugs can be divided into two categories: (i) in situ loading during electrospinning, and (ii) post-electrospinning loading. In situ loading of antibiotics involves electrospinning solutions composed of a polymer and the bioactive compound, or coaxial fabrication of composite nanofibers.

Meanwhile, the post-electrospinning loading of antibiotics is a two-step procedure: electrospun mesh is produced at the first stage, and the mesh is then functionalized by either passive absorption or by covalent linking of bioactive drugs through immersion of native electrospun meshes in solutions of therapeutic molecules [117,118,119]. In general, post-electrospinning loading approaches are alternative methods to incorporate bioactive molecules that decompose under the electrostatic field or that can reduce their therapeutic potential when dissolved. Figure 3 summarizes the primary drug-loading methods, which are suitable approaches for a wide variety of bioactive compounds, including antibiotics.

3.1. Blended Fibers

The basic equipment for electrospinning requires a programmable pump and a feeding system to supply the polymer flow, a high-voltage generator, and a collector. The random orientation distinguishes the most typical assembly of as-spun nanofibers, since manufacturing via a basic electrospinning setup promotes the superposition of subsequent fibers [25], which arise from classical features such as porosity and interconnectivity.

By the electrospinning of polymeric/antibiotic emulsions (Figure 4), different concentrations of antibiotics can be incorporated into the polymeric solution, including mixtures of components. Features such as intrinsic porosity, absorptive capacity, and high surface area made nanofibers an ideal substrate for the design of antimicrobial wound-healing materials [119,121]. The effectiveness of antibacterial polymer/antibiotic composites for drug delivery is driven by the degradation rate of the polymeric matrix and, consequently, by drug diffusion. In this stage, the nature of the polymer and the composite morphology are crucial factors [122].

Following this strategy, Abdoli et al. investigated the incorporation of gum tragacanth (GT), an ideal drug carrier that cannot form nanofibers on its own, into a polyvinyl alcohol (PVA)–graphene oxide (GO) blend, a combination of polymers that possess biocompatibility and drug-delivery properties [121]. A composite fiber of PVA-GT-GO has been prepared, mixed with tetracycline hydrochloride (TCH) and GO, and used to generate electrospun structures for transdermal drug delivery. From this manufacturing, the PVA/GT/TCH and PVA/GT/GO/TCH composites have reached 98% and 82% drug delivery, respectively, at 24 h. During the antibiogram test, both composites have shown bactericidal activity against 12 bacterial strains, with inhibition zones of 23 and 27 millimeters (mm) for E. coli and 25 and 30 mm for S. aureus in PVA/GT/TCH and PVA/GT/GO/TCH, respectively [121].

Similarly, Qi et al. have studied the preparation of poly(lactic acid-co-glycolic) (PLGA) nanofibers loaded with TCH and halloysite nanotubes [122]. In their study, the composite was found to sustain TCH release for 42 days, inhibiting S. aureus growth to a greater extent (80.3%) than the TCH control (81.1%).

Ho et al. have studied the encapsulation of amoxicillin (AMX) in PDLLA before electrospinning composite mixtures or treatment [110]. The composed fibers PDLLA-AMX delivered antibiotics in a sustained manner, inhibiting bacterial growth and reducing periodontal inflammation; moreover, they promoted cellular proliferation along the fiber direction. The in vitro release resulted in a sustained kinetic reaching 81.6% of AMX after 28 days of installation.

Can-Suner et al. used a tricomponent mixture of vancomycin (VAN), ceftriaxone (CFT), and cefpodoxime (CFD) for electrospinning of polylactic acid (PLA) to manufacture wound dressing materials [83]. Regarding the release, the composite produced inhibition halos of 11.66–20.66 mm and 19–27 mm against S. aureus, depending on the CFD and VAN content, respectively. On the other hand, for the E. coli strain, CFT produced superior inhibition, with inhibition zones of 32.33–36.66 mm at concentrations of 5–10%.

Polyvinylpyrrolidone (PVP) nanofibers have been used in combination with the drug metronidazole (MET) for the treatment of bacterial vaginosis due to their mucoadhesive, hydrophilic, and biocompatible properties. In their study, Tuğcu-Demiröz et al. have investigated the bactericidal effect of MET (5 mg/mL), achieving 95% delivery within the first 10 min and an ex vivo permeability flux of 5.4–8.1 μg/cm²h [123].

In this context, electrospinning equipment is particularly well-suited for the fabrication of antibiotic delivery platforms, wound dressings, and other antibacterial biomaterials. In terms of advantages, this type of material is ideal for use as a dressing capable of slowing bacterial growth and promoting cell proliferation due to its mimicry, in terms of structure and composition, of the extracellular matrix at the site of skin injury, avoiding bacterial infection; high interconnectivity and porosity are key factors responsible for these highlights. However, the main disadvantage is the amount of antibiotic loaded into the polymeric solution; depending on its nature (viscosity, dielectric constant, surface tension), a high antibiotic concentration can reduce spinnability, leading to beads and irregular structures. Additionally, the kinetic behavior of randomly oriented fibers can be inconveniently rapid due to the fast degradation rate of the polymeric matrix. Another general consideration is the miscibility of the solvents used, given the polymer and antibiotic solubilities, as heterogeneous structures can result from electrostatic-induced phase separation during electrospinning.

3.2. Co-Axial Electrospinning

The crafting of core–shell nanofibers is commonly achieved via coaxial electrospinning, an approach first reported by Sun et al. in 2003 [124]. In principle, coaxial electrospinning involves the simultaneous processing of two polymeric or composite solutions (Figure 5a) and, with the triaxial setup [125], even three (Figure 5b). The solution supplied in the inner needle is commonly referred to as the core, while the solution provided through the outer needle forms the shell of the final composite fibers. Coaxial electrospinning permits the processing of a spinnable polymeric solution as the shell and a second fluid, spinnable or not, as the core [126]. For producing uniform core–shell electrospun fibers, the critical voltage is a key factor (Figure 5c) [127].

Using the coaxial electrospinning approach, Ali et al. reported the fabrication of coaxial fibers using poly(vinyl alcohol) (PVA)/superabsorbent polymer (sodium polyacrylate) (SAP) embedded with betel leaf extract (BLE) as the germicidal component [128]. In their experimental design, composites comprising PVA/BLE@SAP/BLE/PVA showed inhibition halos of 14–27 mm for S. aureus and 12–24 mm for E. coli, depending on the BEL content of the coaxial fiber core.

Comparatively, Zhou et al. have analyzed the kinetic and antibacterial performance of ciprofloxacin (CIP) loaded on cellulose acetate (CA) nanofibers from blended fibers and coaxial nanohybrids [126]. In their study, CA/CIP coaxial nanofibers reached 78.4% release at 1 h and 90% of the loaded drug after 1.6 h, providing faster release than the one-step method, 38.2% release during the first hour and 90% after 8.6 h., demonstrating that coaxial electrospinning loading improves pulsatile rather than sustained release. In accordance, the in vitro bacterial inhibition produced inhibition zones of 40–54 mm against S. aureus, E. coli, P. gingivalis, and F. nucleatum for the coaxial CA/CIP composite, and 29–44 mm halos for the blended CA/CIP composites.

This electrospinning variation offers several advantages, such as protecting the drug, for example, for the encapsulation of molecules sensitive to organic solvents, increasing their release time, and improving mechanical properties. However, it is a more complex process that requires specialized equipment and is challenging to scale up to an industrial level. Moreover, applying a critical voltage can be challenging for producing uniform core–shell nanofibers.

3.3. Passive Loading

Passive loading of therapeutic drugs occurs due to physical immobilization on electrospun nanofibers, often via sorption [36]. This approach is highlighted for its simplicity in terms of instrumental requirements: it is a two-step method in which the electrospun substrate is fabricated during the first stage, followed by the direct immersion of the as-spun mesh in the drug solution (Figure 6).

According to this protocol, Cheng et al. evaluated the bacterial adhesion onto electrospun polytetrafluoroethylene (ePTFE), collagen, and glycolide fibrous membranes loaded with tetracycline (TTC) and amoxicillin (AMX) [129]. The authors concluded that the loading method does not affect the primary morphology, preserving the microarchitecture to a high degree; however, bacterial adhesion was significantly reduced.

Khampieng et al. loaded doxycycline (Doxy) onto poly(acrylic acid) (PAA) nanofibers [130]. The incorporation of Doxy was carried out by direct immersion of PAA nanofibers in aqueous Doxy solutions of variable concentration (125–1000 μg/mL) overnight. The PAA/Doxy composite released 1600 μg/mL in a sustained manner for 75 days, resulting in inhibition zones of approximately 8, 18, and 20 mm against P. aeruginosa, S. aureus, and S. agalactiae, respectively.

3.4. Covalent and Coordinative Loading

Post-electrospinning functionalization of nanofibrous mats is an alternative that allows overcoming solubility issues and electrostatic decomposition of labile components, thereby enhancing advanced applications [26,27], and antibiotic loading is not an exception [131,132]. Following a two-step approach, electrospinning is the first stage, and antibacterial agent incorporation can be achieved via covalent linking (Figure 7). These post-electrospinning drug incorporations induce modifications in the secondary morphology through chemical bonding. Shi et al. [132] reported the incorporation of metronidazole (MNA) through esterification on PCL, taking advantage of the capacity of cholesterol esterase to hydrolyze the ester bonds selectively, triggering the release of 25.3 μg of MNA per 1 cm² and resulting in bacteria viability of 34.0–36.9%, considering H. pylori as the model of study.

Bueno et al. incorporated Doxy onto electrospun membranes composed of polymer blend (MMA)1-co-(HEMA)1 and (MA)3-co-(HEA)2 [131]. In their study, a polymeric electrospun membrane was treated with a sodium carbonate buffer solution to promote the hydrolysis of carboxyl and ester bonds on its surface, and then Doxy was chemically conjugated. The conjugated membrane prevents bacterial attachment, reducing the colony-forming units (CFU) of biofilm 1.28 × 10⁶ versus 2.59 × 10⁸ in the control at 72 h of incubation.

4. Aligned Nanofibers

Highly oriented fibers are associated with better kinetic performance, enabling sustained release over extended periods compared with randomly oriented nonwoven meshes. Several methods have been reported for producing aligned nanofibers using a special collector system, such as a high-speed rotating mandrel or disks, and parallel electrodes to induce nanofiber alignment [25,133]. The typical set-up variations are presented in Figure 8.

4.1. Mechanical Induction

Using a rotative collector, uniaxially aligned nanofibers can be generated when the collector’s rotation speed is well controlled (Figure 8a) [25,133,134]. Mechanical induction of nanofiber orientation is the most widely used protocol for manufacturing aligned nanofibers for antibiotic delivery. By mechanical induction, Liu et al. produced aligned PCL nanofibers loaded with ampicillin, which produced inhibition halos of 53 and 26 mm in S. aureus and K. pneumoniae, respectively [135]. The kinetic study showed a mixed release mechanism involving small-molecule diffusion and polymer degradation, reaching 95% of ampicillin delivered within 24 h.

Li et al. [28] studied the wound-healing application of composite membranes composed of aligned PCL and aligned polyurethane (TPU) loaded with TCH. Mechanical induction enabled the deposition of aligned patterns capable of releasing 100% of TCH at 120 h and forming inhibition zones of approximately 18 mm in E. coli and S. aureus strains.

Azithromycin (AZ) has also been included in mechanically aligned gelatin methacrylate (GelMa) electrospun composites by Ayoub et al. [136]. In the kinetic monitoring, GelMa-AZ exhibited the highest release capacity (19.4 μg/mL) on day 3. At this time, the antibacterial test showed inhibition zones of 7 and 10 mm for A. naeslundii and A. actinomycetemcomitans, respectively.

Ranjbar-Mohammadi et al. demonstrated that mechanical alignment also benefits the development of antibacterial suture yarns [137]. In their work, they presented the simultaneous electrospinning of poly(vinyl alcohol)/chitosan blend solution and poly(lactic acid) (PLA)/TCH composite solution. The aligned constructions inhibit the growth of E. coli and S. aureus to 20 and 17 mm, respectively.

Nonetheless, the principal disadvantage is often related to the challenge of aligning nanofibers regardless of rotation speed. Another critical concern is the combination of high rotation speed, which can induce fiber fracture.

4.2. Air Gap

The air-gap electrospinning concept was proposed by Li et al. in 2003, resulting in a successful approach for producing uniaxially aligned nanofibers up to several centimeters in length [138]. Since then, much effort has been devoted to increasing the length of aligned nanofibers. However, the length is currently still typically limited to 10 cm [139].

This method consists of arranging two longitudinally parallel electrodes connected to ground. The electrostatic field lines are thus arranged vertically, and the nanofibers charged with opposite polarity to the electrodes, when they reach the gap between them, have no preferred direction in which to organize themselves, since the field distribution exerts equal force on both sides in the direction perpendicular to the electrodes. Therefore, the nanofibers in this area are arranged according to that perpendicular direction (Figure 8b). On the other hand, the deposited nanofibers retain their charge and therefore repel new nanofibers that reach the collector, preventing them from growing thick [25].

In terms of antibiotic delivery, air-gap electrospinning is rarely reported. Ornelas-Guillén et al. proposed the loading of TTC on PVA-aligned fibers following the air gap electrospinning protocol, aided by the choline chloride:urea eutectic mixture [140]. In terms of bacterial inhibition, composites loaded with 23.5 μg produced inhibition halos of 20, 18, and 16 mm against S. aureus, K. pneumoniae, and E. coli, respectively, surpassing the performance of standard TTC disks (30 μg). From the kinetic point of view, the aligned composite undergoes an initial burst, delivering 25–30% of TTC, followed by the sustained release up to 50 h.

However, while air-gap electrospinning has demonstrated multiple benefits for producing aligned nanofibers with superior properties, it presents several limitations that must be carefully considered for its application in antibacterial biomedical environments. These disadvantages are mainly related to aspects of scalability, process control, reproducibility, technical limitations, and potential risks in clinical translation. The main challenges documented in scientific literature are discussed below.

4.3. Centrifugal Electrospinning

Another advanced electrospinning setup is the centrifugal electrospinning. By this method, fiber alignment is promoted by the interaction of the electrostatic field and centrifugal force. In this setup, the polymeric solution is supplied through a rotating spinneret, and the oriented nanofibers are collected in a stationary collector (Figure 8c) [141,142].

Wang et al. studied the layering of aligned PVP nanofibers for the delivery of TCH, achieving at least 98.9% drug entrapment through centrifugal electrospinning [143]. In comparison, randomly oriented nanofibers delivered 87%, monolayered aligned PVP reached 70%, and multilayer aligned composite reached 64% at 5 min. Therefore, the reduced fiber surface area resulting from multiple stacking reduces the release rate—likewise, Hou et al. processed ethyl cellulose via centrifugal electrospinning for the delivery of TCH. After 48 h, the composite delivered 80% inhibition zones of 28.7 mm versus the E. coli strain and 27.3 mm against the S. aureus strain [144].

The highlights of centrifugal electrospinning include its scalability, which is adequate for large-scale fabrication and compatible with a wide range of polymers and composites; in addition, multicompartment spinnerets improve drug loading [145]. However, the intrinsic combination of high voltage and high rotational speed constitutes the most crucial concern from a manufacturing perspective.

5. Patterned Nanofibers

The fabrication of patterned microarrays via electrospinning is primarily associated with near-field electrospinning (NFES). This approach involves performing electrospinning at reduced needle-to-collector distances. Compared with the standard electrospinning setup, the main difference in NFES is the use of X-Y-Z or X-Y mobile platforms to collect the as-spun fibers (Figure 9a), enabling controlled deposition of linear and curved patterns [146,147,148].

Alternatively, 3D printing technology has a significant impact on the manufacturing of patterned electrospun mats, permitting the design and printing of highly organized, complex frames that can be used as collectors to promote nanofiber assembly [149,150].

5.1. Near-Field Electrospinning

Considering NFES as the manufacturing method for antibiotic-delivery scaffolds, Mai et al. reported an NFES configuration for the electrospinning of well-defined patterned composite materials composed of a mixture of polycaprolactone/collagen loaded with the natural antibacterial drug usnic acid (UA) [151]. The well-organized arrangement (produced at 0.4 mL/h, 2.1 kV, 2 mm, and 150 mm/s in hexafluoroisopropanol (HFIP)) exhibited sustained in vitro release of 84% of UA. In their study, it was shown that 1%wt of UA loaded into a PCL/collagen matrix could inhibit the growth of Gram-negative and Gram-positive bacteria, using E. coli and S. thermophilus as model organisms.

Following a similar protocol, Li et al. prepared polycaprolactone-collagen nanofibers loaded with the antibiotic erythromycin (ERY) and analyzed the patterned patches as wound dressings [152]. The composite membranes could prevent the growth of E. coli and S. thermophilus and sustain ERY release at 80%. The bactericidal effect was achieved at concentrations of 1 to 5%wt of ERY, producing inhibition zones ranging from 15.17 to 18.61 mm and from 16.22 to 18.17 mm for E. coli and S. thermophilus, respectively. Moreover, due to the morphological properties conferred by the NFES (produced at 0.4 mL/h, 2 kV, 2 mm, and 80 mm/s in HFIP), the dressing material presented good air permeability and desirable mechanical properties.

NFES patterned assemblies highlighted by high porosity, strong mechanical behavior, and improved aqueous interactions, benefiting their application as DDS. The approach is versatile for electrospinning polymeric composite solutions loaded with antibiotics. However, essential considerations for NFES in the fabrication of drug delivery materials include reducing the length and thickness of the meshes, due to process-specific constraints (e.g., reduced working distance, low flow rate, and highly volatile solvents). Because of the typical reduced needle-to-collector distance, nanofiber fabrication occurs at lower voltages; additionally, highly volatile solvents are required to achieve well-defined nanofiber deposition.

5.2. Patterned Collectors

Using customized 3D printed collectors, Zdraveva et al. reported the fabrication of PCL loaded with cefuroxime (CFU) for dual scaffolding and antibacterial purposes [153]. The 3D-printed collectors allow for tailored topologies, inducing hexagonal arrangements and increasing porosity to up to 90%. Structural features of the electrospun material improved mechanical strength, cell distribution, and confluence compared with random fibers. 3D printed collectors and silicon molds, composed of parallel bars [154,155], perpendicular bars [154], and parallel trapezoidal prisms [156], are reported for the practical fabrication of electrospun mats and are illustrated in Figure 9b–d.

Grgurić et al. compared the use of ribbed and mesh 3D-printed substrates (represented in Figure 9b,c) for the electrospinning of the same PCL/CFU formulation [154]. Both geometries were studied to improve cell adhesion and proliferation. In their study, the authors demonstrate that the geometry of collectors can induce fiber arrangement. Continuing this strategy, Trcin et al. used the ribbed collector for the electrospinning of antimicrobial scaffolding materials based on a PCL/CFU mixture [155]. With the CFU concentration ranging from 5 to 25%wt, the bactericidal effect resulted in inhibition halos of 27 to 40 mm in P. aeruginosa and 31 to 44 mm in S. aureus.

Regarding the drug delivery efficacy, PCL/CFU 5%wt and PCL/CFU 25% could release a maximum of 14% and 9% of CFU, respectively, during the first 4 days; after this period, a slow and sustained release (<10%) was observed, lasting up to 14 days. Moreover, PCL/CFU 5% showed superior performance in limbal stem cell growth and corneal epithelial differentiation; possible cytotoxicity may be related to higher CFU concentrations. The main advantages of using 3D-printed collectors are their rapid, customized fabrication, high compatibility with the electrospinning approach, and the high organization of controllable shapes and geometries induced in the electrospun meshes. On the other hand, the main disadvantage is the specialized equipment required for 3D printing. Other authors also suggest that electrospun nanofibers collected in 3D-printed templates lack sufficient mechanical properties for biomedical applications [149].

6. Hierarchical Structures Combining Electrospinning and 3D Printing

The fusion of electrospinning and 3D printing technologies is a valuable approach to fabricating personalized multilayer materials. From the perspective of antimicrobial resistance, administering antibiotics from multilayered substrates is an innovative alternative that enables overcoming bacterial resistance. The paramount convenience of combining emerging technologies such as 3D printing and electrospinning relies in (i) the versatility of incorporating a wide range of components, (ii) the feasibility of fabricating complex microstructures of high surface to volume ratio and high degree of interconnected porosity, and (iii) the mimicry of extracellular matrix, which is particularly important for the regeneration of infected or damaged tissue [157].

Following this strategy, Song et al. fabricated a bilayer skin scaffold loaded with amoxicillin (AMX) [158]. Their proposed protocol consisted of two stages: the fabrication of a PCL/AMX outer layer by electrospinning and the production of a sodium alginate-gelatine-epidermal growth factor (SA-Gel-rhEGF) inner layer by 3D printing. The resulting bilayer composite PCL/AMX@SG-rhEGF facilitated skin healing while simultaneously preventing bacterial infection; the composite suppressed bacterial proliferation, producing inhibition zones of 2.85 and 3.27 mm against E. coli and S. aureus, respectively. Considering kinetic behavior, the composite released 35.59 μm/mL after 240 h; during the first 24 h, it reached the maximum release concentration (35 μm/mL), thereby establishing sterile conditions in the wound environment. After that lapse, a sustained release remains constant.

A complex bilayer patch PCL-silicon oxide (SiO₂) covered by PCL gelatine methacrylate/sodium alginate and vancomycin (PCL@GelMa/SA-Van), designed to prevent abdominal wall damage, was produced by Hu et al. [159]. At the first stage of patch fabrication, PCL was electrospun; in the second stage, a PCL-SiO₂ coating was deposited to enhance hydrophobicity. Finally, the third stage consisted of performing coaxial 3D printing on the surface of an antiadhesive PCL-SiO₂ layer to construct core–shell-like coatings based on PCL as the core and a mixture of GelMa/SA-Van as the antibacterial layer. The composite patches exerted an antiproliferative effect, reducing the numbers of E. coli (6-times) and S. aureus (4-times) after 24 h compared with the PCL control. The release of Van from composite patches reached 55 μg/mL rapidly after 36 h; thereafter, Van delivery was sustained up to 240 h, preventing post-operative infection in animal models.

In a three-layer wound dressing design, Mirhaj et al. proposed mupirocin (Mup), an alternative antibiotic against methicillin-resistant S. aureus, as the bioactive component added with AgNO₃ [160]. The fabrication of the antibacterial three-layered composite was carried out in three main steps: (i) the construction of the top layer of polyurethane (PU) nanofibrous mesh, (ii) a middle layer composed of pluronic F127-quaternized chitosan-AgNO₃ nanoparticles (F127-QCS-AgNO₃), and (iii) the inner layer composed of core–shell nanofibers of F127-Mup/pectin-keratin (F127-Mup/Pec-Kr). The three-layered composite reached 80% release of Mup at 7 h, producing inhibition zones of at least 3 mm and 2 mm with S. aureus and E. coli strains, respectively.

Using a two-step method, Santos et al. fabricated bilayered membranes for treating periodontitis via a dual-drug delivery approach [161]. A honeycomb substrate composed of polylactic acid/zein/curcumin (PLA/Zein/Curc) was 3D-printed and used as the basis for the electrospun layer. The electrospun coating was achieved by coaxial electrospinning, using a mixture of poly(ethylene oxide)/curcumin/tetracycline hydrochloride (PEO/Curc/TH) as the core and a zein/poly(ε-caprolactone)/β-glycerolphosphate (Zein/PCL/β/β-GP) mixture as the shell. The composite was capable of continuous release of both Curc and TH for up to 8 days, reaching cumulative release of 75% and 65%, respectively, within this time frame. On the other hand, the influence of microstructure on bactericidal capacity was tested; considering PCL/Zein/Curc/TH composites fabricated at different zein concentrations (10, 20 and 30%wt) by electrospinning or 3D printing, as a result electrospun composite exhibited slightly larger (~1.15 fold) inhibition zones than 3D printed composite against clinical strains extracted from periodontal subgingival pocket of chronic periodontitis patients. This result highlights that the superior porosity of electrospun fibers enables higher efficacy in inhibiting bacteria.

Vancomycin and ceftazidime (CAZ) have also been included in multilayer composites produced by coaxial electrospinning on 3D-printed substrates. Yu et al. reported the 3D printing of PCL mesh-like scaffold used for the electrospinning of core–shell poly(lactic-co-glycolic acid)/bone morphogenetic protein (PLGA/BMP-2) and PLGA/Van/CAZ [162]. From the point of view of release, composites released 86% and 84% of Van and CAZ, respectively. By coupling 3D printing and electrospinning, each layer displayed a specific role. First, the PCL mesh fabricated by 3D printing resulted in the structural basis of an artificial bone graft, being the scaffolding component to promote osteocyte healing; secondly, the PLGA/Van/CAZ/BMP-2 electrospun coating prevents bacterial infection due to the sustained release of the antibiotics during a long-term period under in vitro and in vivo conditions.

The combination of 3D printing and electrospinning is suitable for constructing bilayer composites on metallic substrates. Maver et al. deposited two bioactive layer coatings based on carboxymethylcellulose (CMC) and clindamycin (CLIN) on TiAlV and AISI 316LVM surgical substrates [163]. The multilayer composite was fabricated by electrospinning CMC/polyethylene oxide (PEO)/CLIN solutions onto both metallic surgical substrates. Further, six layers (1 mm in total height) of an alginate/CMC and cellulose nanofibrils (NFC) suspension loaded with CLIN were 3D-printed. During the in vitro delivery, the multilayered composites exhibited an initial burst in the first 60 min, reaching 82% and 83% of the CLIN cumulative release after 24 h for TiAlV and AISI 316LVM, respectively.

The combination of 3D printing and electrospinning offers, as a principal advantage, the suitability to achieve complex patterned composites; the precise control of microstructure permits improving mimicry. On the other hand, integrating a wide variety of bioactive components and customizing the structure and properties (bioactive or physical) of each layer are key features that enhance the versatility of this approach. However, the additional cost implied in the 3D printing setup can be considered the limiting factor.

7. General Considerations Regarding Primary Morphology

Unlike randomly oriented fibers, aligned and patterned nanofibers demonstrate better mechanical properties. The production of nanofiber networks with an aligned pattern offers important properties to biomaterials in fields such as tissue engineering, where naturally occurring extracellular matrix biostructures inherently exhibit these anisotropic patterns, including nerve, heart, tendon, blood vessel, muscle, and cartilage tissues. Therefore, the correct architecture of biopolymeric tissues is necessary to mimic the properties of the extracellular matrix correctly [164,165]. On the other hand, patterned and randomly oriented fibers provide interconnectivity and porosity, thereby improving wound-healing capacity [166].

7.1. Equipment Requirements

Beyond the basic electrospinning setup, specialized equipment is required for producing aligned and patterned nanofibers. To promote fiber alignment, additional costs may be considered to install advanced setups such as air-gap, near-field electrospinning, or rotating collectors. The precise configuration of these elements is essential for obtaining aligned and patterned collections with the desired properties for biomedical applications [118,167,168].

Over the last two decades, multiple experimental variants have been developed, ranging from simple designs with two parallel electrodes to complex collectors such as origami, rotating cores, and 3D assemblies, which have enabled exploration of applications in tissue engineering, nerve guides, biomimetic scaffolds, and matrices for the controlled release of antibacterial agents. At the same time, innovative collector designs have been proposed, such as the origami system described by Jha et al. [169], which offers a low-cost alternative for producing highly aligned fibers in far-field conditions. This simplified design avoids the need for high-speed rotation or expensive components, demonstrating that relatively simple geometric modifications to the collector can improve fiber deposition.

7.2. Aligned, Patterned, or Random Nanofibers

Alignment and highly oriented patterning significantly enhance the scaffold’s anisotropy, which is critical for applications such as tissue regeneration (i.e., muscle, nerves, tendons) where cell direction, elongation, and migration depend on the aligned topography of the support. Recent studies have shown that alignment improves targeted cell proliferation, organized extracellular matrix production, and the orientation of collagen fibers secreted by cells, thereby limiting bacterial adhesion or guiding their colonization in a less invasive way when combined with antibacterial agents [118,170].

7.3. Specific Surface Area

Highly controlled frames and aligned fibers tend to have more uniform, often smaller diameters than randomly oriented fibers, resulting in a higher surface area-to-volume ratio. Allowing more efficient functionalization with antibacterial agents (injection, immersion, coating) enhances their controlled release and increases surface exposure for biological interaction and response [170].

7.4. Mechanical Properties and Functional Anisotropy

The orientation of nanofibers contributes to greater mechanical strength, elastic modulus, elongation in the preferred direction, and structural rigidity along the alignment or pattern direction. The orientation is essential for maintaining the integrity of biomedical devices when implanted in areas subject to stress, such as tendons, ligaments, or as coatings for metal implants, where the scaffold must not collapse or deform under physiological forces. In addition, a proper mechanical structure can help resist bacterial attack by reducing deformations that promote fluid or biofilm accumulation in folds [25].

7.5. Permeability, Interconnected Porosity, and Nutrient Transport

Depending on pore diameter, aligned, patterned, or randomly oriented scaffolds can promote the formation of interconnected, more uniform-sized pores, facilitating (at a customized level) the diffusion of nutrients and oxygen and the elimination of metabolic waste. In living tissues, this is critical, as necrosis or bacterial infection can arise in regions where diffusive transport is deficient. Porosity creates virtual “channels” that enhance this transport, improving cell viability, vascular integration, and the effectiveness of diffusion-dependent antibacterial materials, such as sustained release of metal ions. Biomimetic scaffolds with aligned structures and high porosity efficiently support cardiac cell colonization through interconnected pores [118,167].

7.6. Guided Cell Behavior

Cells respond not only to the chemistry of scaffolds, but also strongly to topography and spatial order. In aligned scaffolds, cells such as fibroblasts, nerve cells, and progenitor cells (to name a few) tend to align, which improves their physiological function (i.e., conduction, muscle contraction, nerve regeneration) [164]. On the other hand, skin [171] and bone [172] scaffolds support a higher rate of healing. The orientation can also help reduce unwanted bacterial colonization, or at least guide how biofilm forms, enabling designs to incorporate accessible barriers or antibacterial agents, and even modulating breathability.

7.7. Release of Antimicrobial Agents

Thanks to their ordered geometry, greater surface exposure, and uniform diameters, nanofibrous scaffolds can enable more homogeneous packaging of antimicrobial agents or nanoparticles (e.g., silver nanoparticles, copper nanoparticles, peptide agents) and more predictable sustained release [173]. Homogeneity is essential for antibacterial applications, as irregular release can cause resistance, cytotoxicity, or loss of efficacy. Combining polymers with antibacterial agents in electrospun nanofibers controls morphology, thereby improving release profiles [174].

Electrospun nanofiber morphology, particularly fiber diameter, plays a critical role in determining the degradation behavior of biodegradable polymeric scaffolds and, consequently, the release kinetics of encapsulated drugs. Nanofibers with smaller diameters possess a higher surface-to-volume ratio, enabling more rapid penetration of aqueous media and facilitating polymer chain hydrolysis or enzymatic attack. This increased interaction with the surrounding fluid accelerates polymer degradation, leading to faster drug release profiles than with thicker fibers. For example, studies with paclitaxel-loaded PLGA fibers have shown that submicron-diameter fibers exhibit higher degradation rates and greater drug release than micron-sized fibers, due to enhanced matrix hydrolysis and greater relative surface area. These observations underscore the importance of fiber diameter as a tunable parameter for controlling biodegradation and release dynamics in drug-eluting nanofiber systems [175].

In addition to diameter, other architectural factors such as porosity, core–shell structures, and fiber morphology have been shown to influence degradation pathways and drug release. Tailored porous fibers created via phase separation or coaxial electrospinning can produce distinct degradation environments within the fiber by introducing microchannels or distinct polymer phases that degrade at different rates. For instance, porous nanofibers with core–shell architecture exhibit a synergistic release mechanism in which the outer shell may initially slow the ingress of water, controlling early drug diffusion. At the same time, subsequent matrix degradation governs sustained release over the long term. This interplay between diffusion and degradation control is a central design consideration in fabricating advanced nanofiber carriers for sustained drug delivery, where controlled degradation supports predictable release profiles suited for biomedical applications [176].

Notably, the impact of degradation on drug release is also observed ex situ by comparing different polymer systems under controlled hydrolytic conditions. In electrospun PDLLA systems, the specific alignment between fiber diameter and drug localization influences not only the release rate but also the onset of degradation-mediated release, with smaller diameters favoring quicker onset and larger diameters supporting extended kinetics due to slower matrix breakdown. These results emphasize that fiber dimension is a primary tunable factor not only for diffusion control but also for orchestrating degradation-driven release mechanisms. This insight informs rational design of electrospun nanofibers for antibacterial and other biomedical therapies where time-dependent release is critical [177].

7.8. Compatibility with Multiple Polymers and Functionalization Methods

All electrospinning setups can be adapted to a wide range of natural or synthetic polymers, hybrid blends, copolymers, nanoparticle-containing compounds, and bioactive coatings, among others, allowing the design of customized scaffolds to adjust degradation rate, compatibility, mechanics, and antimicrobial functionalization. Polymers such as PCL, PLA, gelatin, and chitosan have been used to form hybrid nanofibers for wounds, implants, and tissue regeneration [167,178].

7.9. Potential to Reduce Biofilm Formation and Improve Material Hygiene

Although not all studies have directly evaluated biofilm on nanofibers, the high surface-to-volume ratio, porosity, and interconnected structure of nanofibers reduce cumulative surface irregularities, folds, or voids where microorganisms can hide [114,165] compared to non-nanostructured materials. A smoother, more uniform scaffold can be cleaned more easily and, when combined with antibacterial treatments, can reduce colonization. In addition, the localized release of antimicrobial agents from an optimized nanofibrillar structure can achieve greater efficacy with lower doses, reducing systemic adverse effects [179]. Some recent studies of hybrid nanofibers report strong anti-S. aureus and P. aeruginosa activity when controlled release from nanofibers is used [118,174].

7.10. Esthetic Improvements

In biomedical implant or aftercare applications, not only does function matter, but also integration with surrounding tissue, controlled degradation, avoiding permanent residues, reduction in adverse immune responses, and visual surface appearance [180]. Nanofibers can replicate extracellular matrix patterns, giving a more natural appearance, which may promote tissue acceptance. Biodegradable polymers used in aligned scaffolds have shown degradation rates compatible with tissue regeneration [118,167].

8. Equipment and Operating Parameters Considerations

Unlike conventional electrospinning with flat or rotating collectors, advanced electrospinning setups require precise control of nanofiber orientation through the needle-to-collector distance and voltage, depending on the collector features. These requirements increase experimental complexity and error susceptibility, especially in laboratories without prior experience with specialized equipment design. Minimal variation in environmental conditions (humidity, temperature, air flow) can also significantly alter fiber morphology and alignment [118,165,181].

8.1. Limitations on Mesh Length

A recurring disadvantage is that aligned fibers obtained with air gaps or patterned by near-field electrospinning are often restricted in length, which prevents the manufacture of continuous meshes or large scaffolds without additional bonding or transfer processes. This limitation restricts their applicability in the industrial-scale manufacture of medical devices and in coatings for large surfaces, such as prostheses or antibacterial membranes [118,167].

8.2. Limited Thickness

The accumulation of patterned and aligned nanofibers is restricted by the electrostatic repulsion effect between previously deposited fibers and new nanofibers arriving at the collector, and the length of the collector. This results in membranes with reduced thicknesses, which are unsuitable for applications requiring robust three-dimensional structures or high volumetric density, such as bone regeneration implants or multilayer matrices with prolonged release of antibacterial agents [118,170,182].

8.3. Restricted Compatibility with Specific Polymers and Bioactive Fillers

Some polymers with high viscosity, rapid crystallization, or poor stability under high voltage are challenging to process into aligned or patterned structures. Likewise, the incorporation of antibacterial agents (such as metal nanoparticles or antibiotics sensitive to electrical discharge) can be compromised by chemical instability, loss of bioactivity, or non-uniform distribution in fragile fibers [183]. The solubility and dielectric properties may be considered when selecting the most suitable approach and the most convenient antibiotic-loading method.

8.4. Sensitivity to Process Environment

The surrounding air plays a crucial role in electrospinning; variations in relative humidity or temperature directly affect jet stability and the final morphology. In uncontrolled environments, nanofiber formation can result in defective, collapsed, or poorly aligned structures [184] the disadvantages of facilities without environmentally controlled infrastructure.

8.5. Three-Dimensional Integration

Although nanofibers offer advantages in 2D, integrating them into complex 3D structures remains a challenge. The difficulty of stacking multiple layers with stable morphology limits their usefulness in advanced three-dimensional scaffolds that mimic anatomic zones, which are necessary for the regeneration of complex tissues (bone, cartilage, liver). This limitation requires hybrid strategies or combinations with bioprinting techniques to generate true antibacterial 3D scaffolds. However, additional equipment is a prerequisite [185].

8.6. Relatively High Energy Consumption

The need to apply high voltages and maintain stable environmental conditions for prolonged periods can increase the process’s energy consumption [186,187], which is a disadvantage compared to other polymer processing methods that are more efficient in terms of sustainability and cost, which is especially relevant considering the growing demand for green manufacturing and scalable nanofabrication processes in biomaterials [181].

9. Quantification of Antibiotic Release

Quantifying the levels of active compounds in nanofibers is a fundamental step toward understanding their release kinetics, antibacterial efficacy, and overall performance in biomedical applications. Accurate quantification ensures that nanofibers release therapeutic agents at the desired rate and concentration, which is essential for achieving the intended biological effect. To this end, several analytical techniques are employed, each with its own advantages and limitations. Among the most widely used are high-performance liquid chromatography (HPLC), ultraviolet-visible spectroscopy (UV-Vis), mass spectrometry (MS), Fourier-transform infrared spectroscopy (FTIR), and fluorescence spectroscopy [188,189,190,191]. Their principal advantages and drawbacks for quantifying the release of active compounds from nanofibers are summarized in

Table 2. Advantages and disadvantages of analytical techniques towards drug quantification.

Technique	Advantages	Disadvantages
HPLC	High sensitivity, selectivity, and reproducibility; versatile	Expensive, complex, time-consuming sample preparation
UV-Vis	Simple, cost-effective, rapid, non-destructive	Limited sensitivity and selectivity; requires chromophores
MS	High sensitivity and selectivity; provides structural information	Expensive, complex, matrix effects
FTIR	Non-destructive, provides chemical information	Limited sensitivity, quantitative limitations
Fluorescence	High sensitivity, selective for fluorescent compounds	Limited applicability, interference, and photobleaching

.

10. Kinetic Characterization and Modeling Methodologies

The kinetic characterization and modeling of drug release from electrospun nanofibers are essential for understanding the release behavior of antibacterial agents and optimizing their performance in biomedical applications. Release kinetics determine the rate at which active compounds reach the target site, directly impacting their therapeutic efficacy [192,193]. This section provides a comprehensive analysis of methodologies, equations, and computational tools used for kinetic characterization and modeling, with particular emphasis on their application in electrospun nanofibers for antibacterial purposes.

10.1. Methodology for Kinetic Characterization

The kinetic characterization of drug release from electrospun nanofibers typically involves in vitro release studies, in which nanofibers are immersed in a release medium (e.g., phosphate-buffered saline or simulated body fluid) under controlled conditions [194]. Samples of the release medium are collected at regular intervals, and the concentration of the active compound is quantified using analytical techniques such as HPLC, UV-Vis, or MS. The resulting data are then fitted to various kinetic models to elucidate the release mechanism [193]. Figure 10 summarizes crucial aspects in kinetic characterization.

10.2. Kinetic Models

Data obtained from in vitro studies can be fitted to various kinetic models to understand the release mechanism. The most used models include the zero-order, first-order, Higuchi, and Korsmeyer–Peppas models. Each model is based on different assumptions regarding the release mechanism and is described by a specific set of equations [193,194,195]. Equations and generalities are summarized in Figure 11.

The zero-order model describes slow release at a constant concentration and is an ideal kinetic model, in which the drug concentration remains constant during the release process in physiological media and plasma because the release rate does not depend on the drug concentration. The first-order models assume that the amount of drug released over time depends exclusively on the drug concentration loaded into the matrix [196].

The Higuchi model involves two mechanisms responsible for the release rate: swelling and erosion/degradation. Water-solubility and low solubility of a solid or semi-solid matrix promote the drug coverage and decrease the delivery rate [197,198].

The Korsmeyer–Peppas model describes the release mechanism from polymeric nanostructures driven by a Fickian pathway. This model has a diffusion exponent (m) that can be adjusted to distinguish among other mechanisms, while accounting for matrix geometry [199].

10.3. Software Tools for Kinetic Modeling

Kinetic characterization and modeling are essential for understanding the release behavior of antibacterial agents from electrospun nanofibers. Various software tools and add-ons, such as MATLAB, Origin, Excel Solver, R, and Python, provide different advantages and limitations for kinetic modeling. MATLAB and Python offer high flexibility and advanced statistical capabilities but require programming knowledge. Origin and Excel Solver are more accessible and user-friendly, though they may lack flexibility for complex models. R provides a balance between flexibility and accessibility, with strong community support. The choice of tool depends on the specific requirements of the study, including model complexity, dataset size, and the researcher’s programming proficiency [200,201,202].

R is a free, open-source programming language widely used in scientific research for data analysis and modeling. It offers a wide range of packages for kinetic modeling, including nls for nonlinear least squares fitting and deSolve for solving differential equations [200].

Python, also free and open source, is widely used in scientific research for data analysis, modeling, and visualization. Its libraries for kinetic modeling include SciPy for optimization and lmfit for nonlinear least squares fitting [202].

11. In Vitro and Pre-Clinical Studies

In the development of general drug delivery systems, pre-clinical tests are intended to assess safe administration, effectiveness, and dosage efficacy, analyze pharmacokinetic features, and validate non-toxicity. Common preclinical trials include in vitro [203,204,205], in vivo [162,206,207], ex vivo [208,209], and in silico procedures [207,210,211]. The analysis of antibiotic-loaded nanofibers often involves the evaluation of release [123,203,206,212,213] and elution profiles [162,206], bacterial inhibition tests [130,151,205,213], cytotoxicity [121], histologic evaluation [162,214], hemocompatibility [208], immunohistochemical analysis [162], tissue regeneration [119,215], wound healing [49,216], and degradation [204,209]. Figure 12 presents its classification depending on testing conditions. In addition to kinetic analysis, bacterial inhibition and cytotoxicity are among the most widely used approaches for designing nanofibers for antibiotic delivery.

11.1. Bacterial Inhibition Tests

Demonstrating bacterial inhibition is intrinsic to the concept of antibiotic delivery from electrospun nanofibers. The evaluation of the antibacterial activity of nanomaterials requires standardized microbiological tests that allow the effect to be quantified and results to be compared across laboratories. Among the most commonly used methods are the disk diffusion test (Kirby–Bauer), broth dilution tests (micro- and macro-dilution to determine the minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC), well diffusion tests, the E-test method, dynamic contact tests (e.g., ASTM E2149), and standardized tests for textile substrates or surfaces (e.g., ISO 20743, ASTM E2149) [217]. Each technique provides complementary information—inhibition zones, MIC/MBC, time-kill curves, dynamic contact capacity, or activity on surfaces—and its choice depends both on the nature of the material (solid, coating, colloidal suspension) and the expected mechanism of action (ionic release, direct contact, photoactivation) [218].

The Kirby–Bauer disk diffusion assay is a widely standardized qualitative and semi-quantitative method for soluble antibiotics that measures the diameter of the inhibition zone on inoculated agar. Its strength lies in its operational simplicity, low cost, and standardization by bodies such as the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST), which facilitates clinical comparisons. However, it has intrinsic limitations for nanomaterials: diffusion in agar depends on the mobility of the active agent (large nanoparticles do not diffuse), and many nanomaterials exert their effect by releasing ions or through contact interaction, so the absence or small size of an inhibition zone does not necessarily imply a lack of activity. Consequently, disks or wells may underestimate the activity of nanoparticles whose action does not depend on significant diffusion in the solid medium [219]. Broth dilution tests (micro- or macro-dilution) allow the MIC to be determined and, through subculture, the MBC; these quantitative metrics are considered the gold standard for characterizing inhibitory potency. In the context of nanomaterials, microdilution is advantageous because it measures the effect in suspension and therefore captures the activity mediated by released ions or colloidal interactions; it also facilitates the generation of concentration-response curves and calculations of pharmacodynamic parameters. However, the presence of nanoparticles can interfere with optical readings (turbidity) or adsorb dyes/indicators, requiring adaptations (e.g., subculture readings, adenosine triphosphate (ATP) measurements, or alternative colorimetric assays) to avoid false positives/negatives. Therefore, CLSI standards for microdilution are the starting point, but the literature abounds with modifications to manage interference from nanomaterials [220].

Gradient-based methods, such as the E-test, combine diffusion and quantification principles to obtain MICs directly in agar and are widely used in clinical microbiology. However, their applicability to nanomaterials is limited because they depend on diffusion in the solid medium, which is challenging when the antibacterial mechanism requires direct contact or involves slow release of ions with low mobility. Although the E-test can correlate with the MIC obtained by microdilution, its use with nanomaterials requires prior validation and specific controls due to differences in mobility, kinetics, and suspension stability [221].

On the other hand, standardized dynamic contact tests such as ASTM E2149 (Standard Test Method for Determining the Antimicrobial Activity of Immobilized Antimicrobial Agents Under Dynamic Contact Conditions) or ISO 20743 [217] (Determination of antibacterial activity) are better suited for nanomaterials incorporated into surfaces, as they simulate mechanical contact and are less affected by nanomaterial release. They are better suited for nanomaterials incorporated into surfaces, fibers, or coatings, as they quantify bacterial reduction without requiring diffusion through agar. Their strength lies in their practical relevance for solid materials. However, results can vary significantly depending on experimental conditions such as agitation, volume, inoculum, or surface-to-volume ratio, requiring a detailed description to ensure comparability [217].

Complementary methods, such as time–kill, add essential kinetic information for slow-release systems, allowing differentiation between bacteriostatic and bactericidal effects; however, they require a greater experimental workload and strict controls due to optical or colloidal interference inherent in nanoparticulate suspensions, so they are often combined with plate counting [222].

The antibacterial evaluation of nanomaterials presents specific challenges due to inherent analytical interferences, such as light scattering or absorption, dye absorption, and effects on fluorometric readings, which can bias methods based on turbidity or fluorescence. In addition, phenomena such as agglomeration, colloidal stabilization, and the presence of biomolecules can modulate the availability of the active agent, especially in ion-release systems. Therefore, the recent literature recommends combining multiple assays (diffusion, dilution, dynamic contact, time-kill, or biofilm formation) and reporting physicochemical controls, such as size, ζ-potential, or ion concentration, to ensure interpretability and reproducibility and to avoid erroneous conclusions [223].

In operational terms, the Kirby–Bauer disk diffusion assay is rapid and cost-effective; however, it may underestimate the antibacterial activity of materials with limited diffusivity, including nanomaterials and poorly soluble compounds, since its outcome depends on radial diffusion through agar media [224,225]. Conversely, broth microdilution assays allow direct interaction between bacteria and antimicrobial agents, enabling accurate determination of minimum inhibitory concentration (MIC), although these methods can be affected by nanoparticle aggregation and optical interference during turbidity measurements [223,226]. ASTM/ISO standards are suitable for solid surfaces but depend on experimental parameters that affect comparability. In contrast, time-kill and biofilm assays provide kinetic and ecological information, though they require greater experimental complexity. Due to this variability, recent organizations and reviews emphasize the need to harmonize and standardize methodologies by following guidelines from CLSI, EUCAST, ASTM, or ISO, and to rigorously report experimental conditions to enable replication and facilitate comparative analyses for clinical or technological applications [218].

11.2. Cytotoxicity

In addition to the bacterial inhibition assay, the design of antibiotic delivery systems based on nanofibrous mats often involves cytotoxicity analysis. Given the scope of applying nanostructured DDS, cell viability is a crucial aspect. According to ISO 10993-5 and Food and Drug Administration (FDA) guidelines [227,228,229,230], the cytotoxic effects of implantable devices can be assessed primarily using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release, trypan blue, and neutral red uptake [227,228,229,230]. Among them, the MTT is the most widely used protocol for determining cell viability/toxicity in vitro. The effectiveness of MTT assays depends on measuring mitochondrial metabolic activity. This approach refers to the use of MTT. This yellow salt undergoes a color change upon metabolic reduction by the NADH enzyme, yielding the insoluble purple formazan, which is retained by metabolically viable cells [227,228]. Thus, the MTT approach measures mitochondrial integrity, with the amount of formazan proportional to the surviving cell population.

From the point of view of the cell line, rodent and human cell lines are alternatives for cytotoxicity testing, including Jurkat human T-cell leukemia cells, human embryonic kidney HEK-293 cells, human neuroblastoma SH-SY5Ym cells, mouse embryonic NIH-3T3 cells, mouse neuroblastoma N2a cells, and rat hepatoma H4-II-E cells. Nonetheless, the cytotoxicity testing upon cancer-derived cell lines can result in metabolic abnormalities [229].

12. Development of Nanosystems

Over time, pathogenic germs become resistant, particularly to antimicrobial drugs that are overused. Chemists, pharmacists, and physicists conduct research on the development of novel antibacterial agents and on medical education and preventive care. Over the past few years, researchers have focused on the creation and study of polymeric materials with antimicrobial properties to reduce environmental toxicity, and the short half-life typical of low-molecular-weight antimicrobial agents [231]. Enormous efforts to modify existing drugs and design delivery systems have been made over the last three decades, with the sole goal of overcoming antimicrobial resistance. From encapsulation in micelles, the use of polymers, and the control of the micro (nano) architecture to ralentize drug diffusion are some of the tested features for a better therapeutic outcome. Among these nanosystems, electrospun nanofiber meshes represent an advanced evolution, offering superior surface area, porosity, and local retention for topical antibacterial therapy. With advances in combinatorial chemistry and high-throughput screening, most drug candidates selected for development are highly hydrophobic and have negligible water solubility. Because of their poor solubility or slow dissolution in the human gastrointestinal tract, many medications have limited oral absorption. An assortment of formulation methods, including salt formation, complexation, particle size reduction, prodrugs, micellization, and solid dispersions, is now being widely studied in the pharmaceutical field to enhance the oral absorption of poorly water-soluble drugs by increasing their dissolution rates [232].

Metal nanoparticles, emulsion-based nanosystems, nanovesicles, lipid nanoparticles, polymeric nanoparticles, and, recently, nanofiber meshes were among the various categories in which the preclinical safety profile and antibacterial activity of topical nanoformulations were thoroughly examined. Because of their strong antibacterial efficacy and tolerability, topical composites are expected to replace traditional topical formulations in the treatment of skin and soft tissue infections (SSTIs) [233]. Notably, nanofiber-based topical systems provide prolonged residence time and controlled release compared to conventional gels and creams.

As a matter of fact, the drug-delivery electrospun fiber mat is an innovative formulation with potential future clinical applications. Anticancer, antibacterial, and antiviral medicines were used to investigate the effects of drug solubility and compatibility in the drug/polymer/solvent system on encapsulation [234]. Electrospun nanofibers have demonstrated relevance in antibiotic delivery, as exemplified by the following studies. According to Kenawy and colleagues, tetracycline was released from electrospun poly(ethylene vinyl acetate) (PEVA)-PLA (1:1) mats, and PEVA mats provided relatively steady drug release over approximately 5 days [235]. To increase the production of tenofovir (TFV) fibers and assess essential characteristics of the final products, including fiber form, drug crystallinity, and drug loading and release kinetics, Krogstad’s team published a description of the free-surface electrospinning technique in 2014. Employing a nozzle-free manufacturing-scale electrospinning device, PVA containing up to 60 weight percent TFV was effectively electrospun into fibers. The pharmaceutical manufacturing of fiber-based medical textiles for clinical application may be significantly impacted by these discoveries, which establish crucial solution and processing parameters for the scale-up production of TFV drug-eluting fibers by electrospinning [236].

Electrospinning was used to create PCL loaded with ofloxacin (OFL) and PCL: poly(butylene succinate) PBS fibers as a drug delivery method for the treatment of ocular infections. Fiber morphology and size were examined explicitly in relation to several formulation variables, such as the polymer/drug ratio (9:1, 8:2, and 7:3 w/w), solvent systems such as dichloromethane (DCM), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), and dimethylsulfoxide (DMSO), and polymer blends of PCL:PBS at 80:20, 60:40, and 40:60 ratios. P. aeruginosa, S. epidermidis, S. aureus, and E. coli strains served to evaluate the microbiological activity of the modified formulation. Its results clearly showed that freely released OFL from fibers prevented the development of the tested bacteria. The activity of the medication included in the fibers was unaffected by the electrospinning process [237]. This method was employed in recent research to facilitate the incorporation of loaded nanofibers into tablet formulations of the analgesic meloxicam, yielding quick- or sustained-release dosage forms. The tablets can provide the dose in accordance with the anticipated release behavior, and the dominant release mechanism of the tablets was examined using zero-order, first-order, Higuchi, Korsmeyer–Peppas, and Hixon–Crowell kinetics models [238]. According to Soroush and colleagues, mats containing amphotericin B (AMB) and VAN are antibacterial and antifungal agents [239]. Upon the addition of medicines and mucoadhesive agents, SEM images showed a slight reduction in fiber diameter. The Higuchi model was used to fit the in vitro release profile of VAN, whereas zero-order equations were used to fit the AMB release profile. The outcome demonstrated that although other factors influenced AMB release, biodegradable mucoadhesive nanofibrous membranes produced large amounts of VAN within the first 24 h.

In addition to their well-established role as controlled drug-delivery vehicles, electrospun nanofiber matrices can serve as effective platforms for activating prodrugs in response to endogenous stimuli, particularly enzymatic activity and pH changes at infection sites. In this context, prodrugs are designed to remain pharmacologically dormant until they encounter specific biochemical cues characteristic of pathological environments, such as elevated levels of enzymes secreted by bacteria or host immune cells, or the acidic microenvironment typical of infected or inflamed tissues. For example, inflammation-responsive prodrugs synthesized with ester linkages have been successfully integrated into poly(ε-caprolactone)/gelatin nanofibers, where cholesterol esterase secreted by activated macrophages catalyzes ester hydrolysis, triggering the release of the active drug only in the presence of the enzyme; in the absence of enzymatic activity, minimal drug is released, preserving stability and reducing off-target exposure. Such enzyme-responsive strategies demonstrate how nanofiber scaffolds can be tailored to exploit local biochemical triggers, ensuring that the therapeutic payload is liberated predominantly in disease-associated microenvironments, thereby enhancing site-specific activation and efficacy while limiting systemic toxicity [240,241,242].

Beyond enzyme triggers, the pH responsiveness of nanofiber systems enables selective activation of prodrugs in acidic infection milieus. Electrospun nanofiber–hydrogel hybrid dressings have been developed where antibiotic release is governed by pH changes associated with wound infection; these systems remain relatively inert at physiological pH but undergo conformational or chemical changes under acidic conditions, leading to accelerated release of the active agent specifically where bacteria proliferate. This pH-triggered release mechanism is particularly advantageous in chronic or biofilm-associated infections where localized acidity is a hallmark, as it minimizes premature drug liberation and enhances therapeutic concentrations at the site of pathology [243]. Together, enzyme- and pH-responsive nanofiber prodrug platforms represent a versatile toolkit for innovative antibacterial therapy, improving drug stability during delivery, reducing systemic side effects, and maximizing local drug activation precisely where it is needed most.

On the other hand, modifications and repurposing of proven antibiotics, attachment of siderophores, membrane-targeting peptides, and other drug-delivery systems have also been considered adequate vehicles for delivering antimicrobials to pathogenic microbes. Among them, the conjugation of anti-infectious agents to antibodies and nanocarriers has increasingly attracted researchers’ interest [244].

Novel metronidazole analogs, such as ruthenium- and Schiff base-based derivatives, have been developed to address the rise in antimicrobial resistance [245]. These next-generation agents exhibit broader antimicrobial coverage, alternative mechanisms of action, reduced cytotoxicity, improved efficacy against resistant strains, and potential applications beyond traditional infectious disease contexts. Nonetheless, this drug remains the best option for treating Blastocystis infection, even compared with the 1,3-bis-(4-phenyl-[1,2,3] triazole-1-il)2-propanol analog [246].

As repurposed non-antibiotics, phenothiazines enhance the effectiveness of antibiotics by inhibiting efflux pumps. It was noted that thioridazine decreased the ethambutol MIC for M. avium from 8 µg/mL to 2 µg/mL, indicating synergism. According to one study, chlorpromazine decreased the norfloxacin MIC in S. aureus RN4220 from 4 µg/mL to 1 µg/mL, which was interpreted as a combined effect due to efflux inhibition. However, no checkerboard synergic studies were conducted, and FIC values were not determined [247].

In addition to inhibiting biofilm formation and other pathogenicity, the NSAID-antibiotic combination reduced the expression of the efflux pump gene in certain bacterial strains.

As a bio-friendly alternative, loading antimicrobial peptides (AMPs) into topical preparations might prolong their skin contact time. Scalability and affordability encourage the pharmaceutical industry to manufacture and market them; immunogenicity and long-term biological fate, which are the primary barriers to clinical applications; and proper animal disease models and reliable statistical analyses to confirm their therapeutic potential [248]. Synthetic peptide–polymers and their mimics, and in particular structurally nano-engineered antimicrobial peptide–polymers (SNAPPs), are antimicrobial materials with clinical potential as novel therapeutics to combat antimicrobial resistance due to their inherent biodegradability, biocompatibility, and tunable cytocompatibility. Macromolecular design, in conjunction with rational monomer composition, can direct their architecture, self-assembly, and chemical behavior, ultimately guiding the choice of appropriate applications within the biomedical field [249].

In the context of polymicrobial illnesses and biofilms, as with antibiotics and their analogs or metal complexes and NPs, the antibacterial activity of AMP-coated biofilms should be investigated; the materials’ effectiveness should be comparable to or better than that of antibiotics.

13. Regulatory and Scalability Challenges

Although electrospinning has challenges, it stands out for relatively simple adjustments in parameters, namely electrode distance, voltage, concentration, flow rate, and environmental conditions, to reproduce results. Recent studies have shown that even modified setups can enable continuous production of nanofibrous mats at an industrial scale [118,170].

In a biomedical context, technical limitations translate into greater regulatory hurdles for approving devices manufactured using this technique. Morphological variability, the potential cytotoxicity of solvent residues, and the complexity of the process make it difficult to ensure reproducible, consistent clinical standards, delaying the translation of academic prototypes into commercial products in the antibacterial field [187].

The industrial translation of antibacterial nanomaterials poses significant challenges in reproducibility, batch-to-batch consistency, and regulatory compliance, particularly in controlling particle size, surface chemistry, and drug-loading efficiency [250]. These challenges arise from the inherent complexity of controlling physicochemical properties at scale, maintaining reproducibility and uniformity across batches, and navigating regulatory requirements for biomedical applications. For example, reproducibility in size distribution, surface chemistry, and drug-loading efficiency is critical for ensuring consistent therapeutic performance [251,252,253].

Beyond physical and chemical parameters, regulatory and quality requirements for biomedical nanomaterials further complicate scale-up. Regulatory agencies such as the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) require extensive characterization of nanoscale products, including particle size distribution, surface chemistry, sterility, and stability. These requirements are essential for ensuring safety but demand robust analytical methods and validated quality controls that are challenging to implement in high-throughput environments. Compared with other technologies, many nanomedicine products have successfully reached the market, yet this success is often contingent on early consideration of manufacturability and regulatory alignment during development [254].

Cost considerations also play a significant role in industrial translation. High costs of specialty polymers and lipids, combined with the need for specialized equipment and energy-intensive processing, can render specific nanomaterial manufacturing strategies economically unfeasible without further process optimization. Continuous manufacturing approaches are being explored to reduce operational costs and improve scale-up feasibility by enhancing control and reducing waste. These continuous systems can achieve consistent product quality, but they introduce new engineering challenges related to system integration and control stability [255].

Finally, environmental, health, and safety (EHS) concerns associated with large-scale nanomaterial production must be addressed during industrial translation. The use of volatile organic solvents and the potential use of nanomaterials in the environment necessitate comprehensive lifecycle assessments (LCAs) and implementation of solvent recovery and waste management strategies. Moreover, the long-term impacts of engineered nanostructures on human health and ecosystems remain an active area of research, underscoring the importance of sustainable manufacturing practices that minimize emissions and exposure risks throughout the product’s life cycle [250].

Likewise, translating electrospinning technologies from laboratory-scale research to industrial nanomanufacturing remains a significant challenge. Although electrospinning is widely recognized as a versatile technique capable of producing fibers with controlled diameter and morphology, conventional capillary systems suffer from intrinsically low throughput, typically in the range of 0.01–1 g h⁻¹, which severely restricts their industrial implementation [256,257]. This limited productivity is insufficient for biomedical devices, functional textiles, and filtration applications that require mass production [183]. Moreover, technical limitations related to jet instability and process reproducibility further complicate scale-up [258,259].

One of the principal challenges in industrial translation is balancing productivity and product quality. Increasing production rates often compromise fiber uniformity and morphology due to factors such as inadequate solvent evaporation, jet instability, and interactions among multiple jets in multi-needle setups. For example, while multi-needle electrospinning configurations have been developed to increase production capacity, electrostatic field interference between needles can result in inconsistent fiber deposition and morphological defects, ultimately affecting the reproducibility of fiber properties. Additionally, needle clogging remains a persistent operational issue, which not only disrupts continuous operation but also increases maintenance complexity and costs [260].

Alternative strategies to address scale-up limitations include free-surface and needleless electrospinning, in which multiple jets are generated from an open liquid surface without discrete spinnerets. These approaches can enhance output by enabling the simultaneous formation of several jets; however, they introduce challenges, such as increased demands on process control, higher voltage requirements, and issues with solvent management under high-throughput conditions. Free surface systems, while mitigating clogging, often require precise control over environmental factors such as humidity and temperature to maintain fiber consistency, thereby increasing equipment and operational complexity [260].

Another significant consideration for industrial nanomanufacturing is environmental, health, and safety (EHS) compliance. Many electrospinning processes rely on volatile organic solvents for polymer solution preparation, which present ecological and occupational hazards at larger scales due to solvent vapor emissions and worker exposure unless effective recovery and mitigation systems are implemented. These environmental concerns, including residual solvent toxicity and regulatory VOC compliance, are emerging barriers to large-scale adoption of electrospinning technology [186].

Additionally, the high voltage required for electrospinning poses operational safety risks, necessitating rigorous insulation, grounding, and control systems to protect personnel and equipment from electrical hazards.

Environmental control and process parameter sensitivity (e.g., temperature and humidity) also influence fiber morphology and consistency, posing challenges for maintaining uniform fiber diameter distribution and nanostructure quality during scaled-up production. To address some of these concerns, green electrospinning strategies using biologically benign solvents have been proposed to reduce solvent toxicity and environmental impact, representing a promising step toward safer, scalable production systems [261]. Quality control and consistency across large production batches are essential for end-use applications such as drug delivery systems, wound dressings, or filtration membranes; therefore, advanced process monitoring, environmental control, and real-time feedback systems are necessary to adjust parameters and detect deviations in fiber morphology dynamically, but such technologies are still under active development [259].

Additionally, achieving cost-effective scalability requires not only equipment innovation but also process standardization and integration with downstream manufacturing steps. For nanofiber applications in pharmaceuticals, for instance, integrating electrospinning with sterile filling, coating, and packaging processes under Good Manufacturing Practice (GMP) conditions represents a substantial challenge that extends beyond fiber production itself. There are relatively few documented industrial case studies in the literature that demonstrate end-to-end solutions from polymer dissolution to finished biomedical products, underscoring the need for collaborative efforts between academia, industry, and regulatory bodies to fill this gap [262].

Emerging approaches seek to mitigate these obstacles through innovative spinneret designs, the incorporation of secondary forces (e.g., centrifugal and magnetic forces), and the development of hybrid processes that combine electrospinning with complementary manufacturing techniques such as centrifugal spinning or blow spinning. While these strategies show promise for enhancing throughput and fiber quality, further research is needed to validate their scalability, cost-effectiveness, and compatibility with regulatory requirements for biomedical nanofiber products. Consequently, the full industrial potential of electrospinning remains constrained by technological, economic, and regulatory challenges, and realizing this potential will require sustained innovation in nanomanufacturing science and engineering [263].

In summary, the successful industrial scaling and commercial translation of antibacterial nanomaterials requires a multifaceted approach that integrates process engineering, quality control, economic analysis, regulatory foresight, and environmental stewardship. While laboratory-scale research continues to innovate new compositions and functionalities, bridging the gap to industrial production remains a critical challenge that demands collaboration across disciplines and sustained effort to align technological innovation with practical manufacturing realities.

14. Progress and Potential

The incorporation of antibiotics into nanostructured polymeric materials is a promising alternative for overcoming bacterial infections. The suitability of electrospinning for the versatile fabrication of polymeric nanofibrous templates stands out. Moreover, variability in antibiotic loading via different approaches improves kinetic and pre-clinical antibacterial performance. Bactericidal or bacteriostatic activity can be modulated by combining the loading mechanism with the structural features of electrospun meshes, demonstrating the versatility of electrospinning as a manufacturing tool for antibiotic release and other antibacterial aims. The principal advantage of combining nanofibers and antibiotics lies in the enhanced efficacy of bacterial inhibition enabled by nanostructured platforms.

Considering comparative aims, efforts to adopt conventional methods and ensure reasonable discussion are required, especially regarding inhibition results and release efficacy (i.e., mm versus Colony-Forming Units (UFC) and μg/mg versus %, respectively). The use of standardized guidelines and procedures orients this aspect, but there is still a long way to go.

Currently, there is no solid clinical evidence demonstrating that antibacterial nanoformulations (including nanofibers, metallic nanoparticles, or polymeric nanoparticles) are directly associated with emerging infectious diseases or induced pathologies in humans. Most available data come from in vitro studies and animal models, which mainly evaluate antimicrobial efficacy and toxicity, without long-term epidemiological follow-up. Systematic reviews on nanomedicine indicate that clinical evidence remains limited, as many of these technologies remain in preclinical phases or in exploratory trials. In the case of nanofibers for wound healing, studies report a significant reduction in bacterial load without clinical adverse effects attributable to the material, supporting its preliminary safety.

However, experts warn that the extensive use of nanomaterials in consumer products and in uncontrolled environments could expose human and environmental microbiomes to sublethal concentrations, favoring bacterial selection. Although there is still no direct clinical evidence, environmental studies suggest co-selection of genes conferring resistance to both antibiotics and nanomaterials, posing a potential long-term concern and, at the same time, offering a research opportunity.