Antimicrobial Effect of Electrospun Nanofibers Loaded with Silver Nanoparticles: Influence of Ag Incorporation Method

Facultad de Ciencias de la Ingeniería y Tecnología, Universidad Autónoma de Baja California, Unidad Valle de las Palmas, Blvd. Universitario 1000, CP, 21500 Tijuana, Baja California, Mexico Facultad de Ciencias Químicas e Ingeniería, Universidad Autónoma de Baja California, Unidad Otay, Universidad #14418, UABC, Parque Internacional Industrial Tijuana, Tijuana, Baja California, Mexico Centro de Nanociencias y Nanotecnología, Universidad Nacional Autónoma de México, Km 107, Carretera Tijuana-Ensenada, CP, 22860 Ensenada, Baja California, Mexico Department of Technology of Organic Substances and Polymer Materials, Tomsk Polytechnic University, 634050 Tomsk, Russia Research Department, Sevastopol State University, Sevastopol, 299053, Russia Research Institute of Clinical and Experimental Lymphology-Branch of the Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, Novosibirsk, Russia Escuela de Ciencias de la Salud Unidad Valle Dorado, Universidad Autónoma de Baja California, Ensenada 22890, Baja California, Mexico


Introduction
In the last decades, the interest of the biomedical industry in nanomaterials has increased due to its promising applications against different diseases. In this sense, AgNPs are among the most studied nanomaterials principally due to their highly efficient antimicrobial properties [1][2][3][4][5][6]. It is known that AgNP efficiency increases using a carrier [4].
Electrospun nanofibers are ideal carriers for AgNPs since their small dimensions permit homogeneous distribution and avoid mass aggregation. The blend of technologies between nanofibers and nanoparticles maximizes both structure properties, making them an ideal amalgam for many applications.
Thus, this amalgam of nanofiber/AgNP system properties has been proved and attributed to the metallic nanoparticle proportion, size, and spatial distribution of AgNPs in the fibers [42] (entry 3).
A comparative study of AgNP loading methods on PVA was performed by electrospinning AgNO 3 -polymeric solutions followed by a reduction posttreatment. In that study, the AgNPs were mixed in the polymer solution before the electrospinning process, and the resulting polymeric scaffolds were immersed in a silver solution followed by a reduction process (comparing UV and thermal treatments). The authors reported that UV reduction was the more efficient method to incorporate AgNPs on nanofibers' surfaces [49].
In this work, we compare the results published regarding the incorporation of AgNPs in nanofibers, including all the above methods. Nevertheless, AgNP-loaded nanofibers differ in their activity depending on the silver loading method into the nanofibers. Hence, this review compares different techniques of AgNP incorporation into electrospun nanofibers and their antimicrobial effectivity to identify the most promising method and discuss the current limitation of each of them.

Methods of AgNP Immobilization for Loading of Electrospun Nanofibers
In literature, several strategies merge the electrospun nanofibers and AgNPs, which coat the nanofibers' surface or are embedded into the bulk [50].
The parameters considered in this work to determine the best among the discussed methods include the AgNP distribution in/on the electrospun fibers. Bortolassi et al. discussed that the bioactivity of the AgNPs depends on their capacity to attach to the microbial cell membrane's surface, altering the permeability and cellular homeostatic, thereby AgNP distribution and availability over the surface of the fibers become crucial for the bioactivity. Hence, the combination of the high specific surface area of the electrospun fibers and an AgNP high loading with homogeneous distribution over the surface of the fibers become a desirable design for the high final antimicrobial activity [51,52]. For instance, the loaded AgNP nanofibers fabricated by the direct addition of AgNPs into the polymeric solution decreased the antimicrobial efficiency of AgNPs due to their aggregation. When the AgNPs are incrusted in the fibers, they are not exposed for direct contact with the cellular membrane [53].
Also, it is remarkable that the presence of AgNPs into/on the polymeric nanofibers affects its intrinsic properties. The most important characteristic that is modified is its bioactivity, which is improved [46,[51][52][53]. Among other properties that are affected by the presence of the AgNPs on/in the fibers are the mechanical properties such as reduction in surface tension [54], increase in average fiber diameter [36], and changes in thermal properties including the glass transition temperature, degradation temperature, and temperaturedependent mass loss. In general, AgNP-loaded nanofibers are more resistant to heat. All property changes mentioned above are caused by the structural changes of the polymeric backbone [36,46,47].
Below, we present and compare several methods to create AgNP-loaded electrospun nanofiber scaffolds.

Direct Blending Method.
A facile method to produce AgNP-loaded nanofibers is the direct blending of premade AgNPs in the polymer solution before electrospinning [35], being preferred the AgNP colloidal solutions for easy incorporation into the nanofibers [55]. The encapsulation of AgNPs within the poly(έ-caprolactone) (PCL) microfibers without Ag at the surface of microfibers allows controlling release of AgNPs from the hybrid constructs in combination with high antibacterial activity [56].
Several polymeric systems have been reported to be electrospun and loaded with AgNPs, such as the case of poly(vinylidene fluoride) (PVDF) [20] (entry 4), PVA/poly(urethane) (PU) [57] (entry 2), nylon [42,58], and poly(vinyl pyrrolidone) (PVP) [59] (entry 1), among others. This versatility of modified nanofibers is because AgNPs can be prepared with various solvents such as formic acid (FA), dimethylacetamide (DMAc), water (H 2 O), and hexafluoro propanol (HFIP). Also, it has been recognized by several studies that the loaded amount of AgNPs can greatly differ from 0.1 to 30% wt., of the total polymeric mass. On the other hand, it has been observed that the higher the content of AgNPs added to the polymeric solution, the higher the conductivity, promoting smaller fiber's diameters [35,55]. These resultant fiber morphologies can be rough due to the coating and immersion of AgNPs mentioned before [55].

Experimental Conditions of the Direct Blending
Method. For a reported direct blending technique, the first step was the dissolution at 50°C of nylon 6 (15% wt.) in formic acid, a reduction agent for AgNO 3 . In this study, the AgNO 3 (0.5 and 1.25% wt.) was slowly added to the polymer solution, kept in darkness with constant stirring at room temperature for 24 h, enough time for the reduction of 2 Journal of Nanomaterials AgNO 3 . After that, the final dispersion was electrospun [42] (entry 3).
In another study, tannic acid prepared in an ammonia solution (NH 3 ·H 2 O) was added to the polymer solution as a reducing agent of AgNO 3 . After one hour, the powder obtained by solution concentration was resuspended in acetone and filtered. Synthesized AgNPs were extracted using vacuum drying at room temperature and then added to a PVP solution before electrospinning ( Figure 1).

UV-Irradiation
Method. The amount of AgNPs loaded on nanofibers is more significant when a UV-irradiation method is used to produce the initial silver burst release, either in the form of AgNPs or in the residual Ag + ions. This method promotes incorporating the AgNPs into nanofibers' surface in a random distribution [44][45][46]. The method induces the silver ion migration from the core to the surface during the formation of the AgNPs [45]. Also, it has been described that the UV-irradiation method achieves smaller AgNP diameters with narrower distributions than the other methods. These results are relevant for antimicrobial activity because the high surface area of AgNPs promotes a faster Ag + ion release [60] (entries 8 and 16).
Moreover, the biological activity of electrospun scaffolds is related to the AgNO 3 content. It was reported that AgNP-loaded poly(ether amide) (PEBA) fibers were fabricated using 0.15% of AgNO 3 in the polymer solution for electrospinning inhibits >99.99% of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) cultures. However, when the AgNO 3 concentration decreased (0.05% AgNO 3 ), the bacterial inhibition decreases (20% less activity in both bacterial strains) [61] (entry 7). A similar activity was observed on loaded AgNPs in gelatin nanofibers evaluated against S. aureus and E. coli, with selective activity for the later strain. Authors suggest that the different biocidal effect is due to the generous peptidoglycan layer next to the cell membrane of Gram-positive bacteria such as S. aureus, which serves as a protective structure for external threats reducing the AgNP introduction to the bacterial cell [60].
Phan et al. [46] (entry 5) synthesized silver/polyacrylonitrile (Ag/PAN) nanocomposite membranes testing their antibacterial activity. In this study, electrospun AgNP/PAN nanofibers were prepared from the solution of PAN and AgNO 3 using the UV-irradiation method to reduce the Ag + ions into AgNPs. The nanofiber antibacterial activity was tested against E. coli and Bacillus subtilis (B. subtilis), finding long-term bactericidal effects. The authors claim that these nanofiber systems are useful for water purification, bacterial filtration, and biomedical devices [46] (entry 5).

Experimental Conditions of the UV-Irradiation
Method. In the case of the UV-irradiation method, most of the studies prepared the Ag-loaded nanofibers by mixing the polymeric and AgNO 3 solutions previous to the electrospinning process [44,45,[60][61][62] except for Phan et al. [46] (entry 5) that electrospun the fibers before the AgNO 3 incorporation. An advantage of this method is that, as reported, no extra time or additional solvent is required [44][45][46][60][61][62][63]. Besides, no further treatments are needed after the irradiation step, and the exposition time to UV light can be changed depending on the desired results. Interestingly, it has been reported that after four hours of irradiation, the number and size of the AgNPs continuously increased [44]. Figure 2 explains the UV-irradiation method general procedure, where the incrusted AgNPs can be appreciated over the nanofiber surface.
Additionally, the electrospinning conditions in both methods (direct blending and UV irradiation) are selected depending on the polymer system used [62]. After the electrospinning process, the UV irradiation step is developed. Here, the UV light's specific wavelengths are 254 and 365 nm [46]

Silver Mirror Reaction Method (SMR).
The SMR method is a versatile method that uses different substances as reducing agents for AgNO 3 solution to produce the AgNPs, which coat the surface of an object submerged in the reaction solution. This reduction can generate a visual phenomenon that It is used to create a controllable smooth coating over different surfaces: for large surfaces (e.g., telescope glasses) and extremely small surfaces (nanofibers) [64] (entry 13). The reaction occurs at ambient conditions, which is very appropriate for nanotechnology applications [65] (entry 11). Even though using this method is impossible to achieve shape control of AgNPs, the technique can produce quasispheres, wires, rods, right bipyramids, beams, spheres, cubes, or octahedrons [65,66].
Several applications have been reported for SMR synthesized nanofibers. Excellent antimicrobial activities against bacteria and fungi were observed with PAN/AgNP nanofibers with AgNPs evenly dispersed on the nanofiber's external surface. Those were prepared with PAN nanofibers pretreated in AgNO 3 aqueous solution followed by the SMR process [48]. Also, noble metal nanoparticles loaded on electrospun nanofibers have been synthesized due to their potential use as sensors [67]. Another application is for silver nanowire membranes, in this case, Ag + ions coated poly(acrylonitrile-co-phenylethylene) (P(AN-S)) nanofibers used as a template for the reduction of a silver solution on the nanofibers' surface. The SMR process allows silver deposition on the nanofiber's surface. Thus, nanowires were developed and characterized by scanning electron microscopy (SEM), energy dispersive spectrometry (EDS), and X-ray diffraction (XRD). These nanowires have different resistance and transmittance on PET and glass: 15 Ω/sq and 80%, and 37 Ω/sq and 81%, respectively [65].

Experimental Conditions of the SMR Method.
The reported methodologies differ in time and posttreatment steps [53,54], but an advantage over other methods is that the reaction can be made at room temperature. In two manuscripts, the polymeric solution needs a 12 h reaction at room temperature before the electrospinning process [48,65]. In another study, just 30 min were used, and a sol-gel process was done before the electrospinning step [64]. Hence, the polymer solution preparation time is not standardized and differs depending on the methodology used to achieve complete polymer dissolution. Wang et al. prepared PAN nanofibers stirring the polymeric solution just for 1 hour at room temperature, using the same polymer as the compared stud-ies. Hence, these polymeric solution reaction times can vary depending on the polymer chosen and the AgNP incorporation method [11]. Within the reported procedures, we can find nanofibers exposed to the AgNO 3 solution for 18 and 24 h in a dark room at room temperature [48], and AgNO 3 solution mixed with the polymer solution before the electrospinning process [64,65]. Hence, the AgNO 3 solution can be added before or after the formation of fibers.
Regarding electrospinning, parameters depend more on the polymeric solution than on the AgNP immobilization method or the presence of AgNO 3 . The electrospinning parameters determine the morphology, diameter, porosity, and distribution of the fibers [20]. After fiber preparation, several posttreatments are needed. Obtained fibrous mats are submerged into AgNO 3 solution at the desire concentration, usually in a 1 : 2 AgNO 3 /polymer solution ratio, but other relative proportions AgNO 3 /polymer were also tested [64,65]. Ag + ions are deposited over the fiber's surface when the AgNO 3 solution is added to the fibers earlier prepared. In contrast, Ag + ions get encapsulated into the fibers if AgNO 3 solution is added before fiber preparation [68]. Other procedures include fibrous scaffolds freeze-drying before the SMR [48] or a two-step filtration with an organic-free filter followed by a 0.2 μm filter to removed particles [64].
Other differences were observed for the reported SMR step. In some cases, the authors dropped ammonium hydroxide (NH 4 OH) into AgNO 3 solution to prepare a diamine silver (I) (Ag (NH 3 ) 2 + ) solution [65] (entry 11). Others used a hydrazinium hydroxide (N 2 H 5 OH) solution as a reducing agent [64] (entry 13). On the other hand, Shi et al. [48] (entry 12) submerged the fibers into the AgNO 3 solution, then added concentrated ammonia (NH 3 ) (2.5% wt.) into the beakers just until the brown precipitate dissolved, and then added formaldehyde (HCHO) (1% wt.) as the final step. Additional steps after SMR as a threefold washing procedure with distilled water or 40°C vacuum oven drying are reported [64,65].
A simplified schematization of the silver mirror reaction method showing the AgNP distribution on the nanofiber's surface is represented in Figure 3.

Thermal Reduction
Method. This method is promising due to the easy steps to perform. Some advantages of this method are as follows: uniform AgNP distribution on the   Journal of Nanomaterials nanofiber's cross-section [69], decomposition temperature decreases when Ag content increases on PVA-AgNPs [49,62], and more desired stability (less probability of AgNP aggregation). However, AgNP leaching could inactivate the membrane once the first Ag + release from the nanofiber surface has occurred [62]. Silver conducting nets were synthesized using the thermal reduction method over a plane scaffold prepared with electrospun nanofibers of poly(methyl methacrylate) (PMMA), and silver trifluoroacetate (STA) deposited on transparent substrates. Those nets had high transmittance and low resistance [70]. STA is reduced to AgNPs with a 100°C postthermal treatment that decomposes the organic polymer to achieve the one-dimensional net configuration. The nets' sheet resistance was as low as 15 Ω/sq correlated with the morphology and the STA/PMMA ratio. Properties as surface plasmon resonances (SPRs), fiber morphologies, and electrical and optical properties (diffusive optical transparency of ≈54%) were determined. For the electrospinning technique, no special modifications were added. The optimization of the fiber formation and the electrospinning parameters were chosen depending on the properties (viscosity, concentration, conductivity, and surface tension) of the polymer solution [20,47,62,70].
CA fibrous mats were synthesized with a simple alkaline treatment (NaOH) by submerging the fibers in the solution for 48 h, creating cellulose fibers (CEF). The CEAg samples were obtained immersing the CEF for 24 h at 23°C in an AgNO 3 solution then dried for 2 h. Finally, the reduction process of CEAg was done in a drying oven at 160°C for 1 h, 1.5 h, and 2 h [47]. Silver nets also could be obtained by thermal decomposition at 500°C of PMMA for 3 h under air or nitrogen atmosphere. In this case, AgNPs were obtained by reduction of silver precursors for 12 h at 100°C [70]. Contrary to the previous two discussed methods, before the electrospinning process, Lin et al. [62] (entries 8 and 16) pretreated the polymeric/AgNO 3 solution at 105°C for 1 h, no further treatments after the obtention of the fibers. Figure 4 represents the general procedure of the thermal reduction method.
AgNP particle size and average nanofiber diameter obtained with four AgNP incorporation methods are presented in Table 1.   6 Journal of Nanomaterials

Antimicrobial Activities of Ag Nanofibers
AgNPs are well known to have antimicrobial bioactivity. This bioactivity occurs through cell membrane damage, free radical generation, and DNA interaction, among others ( Figure 5) [71,72]. One of the proposed mechanisms of AgNP action is the effect on the lipid bilayer induced by the AgNP accumulation in the bacterial cell wall [59]. Thus, membrane permeability increased, causing cell damage and death. Moreover, the effect increases while the AgNP size decreases [73]. Another mechanism suggested for cell death induced by AgNPs is the reaction with thiol groups (-SH) of cysteine and phosphorus compounds on the cell wall, affecting respiration and replication processes [59,74]. Another explanation for the antimicrobial activity is that metal depletion may cause the formation of irregularly shaped pits in the outer membrane and change membrane permeability, which is caused by a progressive release of lipopolysaccharide molecules and membrane proteins [63] (entry 6). AgNPs or the released silver ions (Ag + ) can also enter the bacterial cells and interact with compounds containing sulfur and phosphorus, preventing DNA replication and inactivating proteins. Besides, they can inhibit the activity of endocellular ATP levels, thereby preventing the cell's respiratory function. Furthermore, AgNPs have been reported to induce the release of reactive oxygen species (ROS), forming free radicals with strong bactericidal effects ( Figure 5) [75]. AgNPs have a broad antibacterial spectrum covering aerobic, anaerobic, and Gram amphoteric bacteria [76], low incidence of resistance [77], and sustained antibacterial activity [78], thus have been widely used in antibacterial wound dressings [75].
The antimicrobial activity of AgNPs depends on the surface area of the nanomaterial [72]. The highest concentrations of released Ag + ions have been observed from AgNPs with the highest surface area. On the contrary, Ag + ion's low release has been found for AgNPs with low surface area, resulting in weak antimicrobial properties [79]. Most electrospun nanofibers do not affect microbial cell reproduction by themselves, but only with the presence of AgNPs. Such is the case of PAN nanofibers which were endowed with excellent antibacterial properties due to the introduction of AgNPs. The authors claim that AgNPs have strong antibacterial properties since they attach to the cell walls and disturb cell-wall permeability and cellular respiration [48] (entry 12).
Poly(vinyl alcohol-co-vinyl acetate)/octadecyl aminemontmorillonite) (P(VA-co-VAc)/ODA-MMT) nanofibers loaded with AgNPs showed high antimicrobial activity against fungus (Candida albicans, tropicalis, glabrata, keyfr, and krusei) and bacteria (S. aureus and E. coli) [80]. It has been demonstrated that Ag + release confers the AgNP microbicidal effect [81][82][83]. Moreover, the internalization of AgNPs into the fibers permits a longer bioavailability of the silver on the application site because the release of the A + ions depends on the time of fiber degradation [84]. This could explain that 10 nm AgNPs were more toxic for E. coli than 20-80 nm due to a more efficient cell-particle contact. Thus, AgNP toxicity correlates with size and content because of the Ag + initial release rate, affecting the fibrous scaffold cytotoxicity. However, the AgNP amount also regulates the long-term Ag + release rate and, therefore, the microbicidal activity [72,75].
AgNP-loaded electrospun membranes with antibacterial properties have been tested in food packaging material to delay food spoilage or bacteria contamination [63,78]. Chaudhary et al. [63] (entry 6) used an electrospun AgNP/-PAN composite filter media to cover a nutrient media in room conditions and pass ambient air through the filter media. The nutrient media protected by the nanofibrous filter remained free of bacteria growth after two months, while the unprotected nutrient media show microorganism growth. Taking advantage of the excellent dispersion of AgNPs in electrospun nanofibers, Castro-Mayorga et al. [85] reported electrohydrodynamic processing, which combines the electrospraying and electrospinning techniques to produce a multilayer system comprising a poly(hydroxy alkanoate) (PHA) substrate and an electrospun PHA coating containing AgNPs. The materials reduced the Salmonella enterica population below the detection limits at a very low silver loading of 0.002% wt. Table 1 shows the physicochemical and antimicrobial properties of AgNP-nanofiber systems prepared by the discussed methods. It can be observed that each of the four methods affects the resulted size of the incorporated AgNPs. Moreover, small particle sizes (<10 nm) can be achieved in all methods.
It should be noted that the data in Table 1 include the following: (1) inhibition zone diameter, which may not be comparable, or (2) results of distinct AgNP concentrations leading to approximately 100% inhibition of microorganism growth, which is also not representative. In the future, the minimal inhibitory concentration (MIC) and the inhibitory concentration (IC 50 ) should be presented as data; moreover, for this purpose, the microdilution test to evaluated antimicrobial activity should be used [37].
Also, it can be appreciated that information about the resulting AgNP size is not reported in some articles. The antimicrobial results are reported in different ways, making it difficult to compare studies.

Comparisons among Methods of Incorporation of AgNPs
Metal nanoparticles tend to aggregate in the polymer matrix during nanofiber formation, highlighting the need for new and better methods that allow better metal nanoparticle dispersion on the polymer nanofiber matrix [42] (entry 3).
Another requirement is to reduce the number of reaction steps and reduce the use of toxic chemical agents to form AgNP/composite polymeric scaffolds. The search for a facile and ecofriendly method is an important task [86]. There is not a noticeable difference in AgNP size and antimicrobial activity for the studied methods; they all reported similar results (Tables 1 and 2). Hence, in our further discussion, we can focus on finding the method possessing the most advantages other than appropriate AgNP size. Among the compared methods, the direct blending method is the easiest, more accessible, and most commonly 9 Journal of Nanomaterials used technique to incorporated AgNPs into nanofiber mats [35,55]. It has been reported that the electrospinning conditions do not affect AgNP bioactivity on microbial cells; however, fiber-matrix encapsulation could reduce it [87]. Despite that, the lack of chemical treatment, irradiation, and thermal treatment made the direct blending method preferable compare to others that consume more time and energy [42] (entry 3). However, it has been described that the mechanical mixing of the AgNPs with the polymeric solution by the direct blending method affects the homogeneity of particle dispersion, facilitating their aggregation, increase viscosity of the polymeric solution, which increase the surface tension, complicating nanofiber formation [66] (entry 13).
Otherwise, comparing the UV-irradiation method with the direct blending, an initial burst release of the AgNPs incorporated into the nanofibers is observed only in the UV method. As mentioned above, this burst release was attributed to the silver ion's migration from the core to the nanofiber surface [45]. Moreover, in the UV-reduction method, the AgNP diameters are smaller with narrower distribution than other methods. In its turn, the size of AgNPs is an essential parameter of antimicrobial activity [62] (entries 8 and 16). AgNP/PVA nanofibers prepared by the UV irradiation method showed more effectivity against S. aureus than those prepared by the thermal reduction method. In the first case, after 30 minutes of incubation, no bacterial colonies were detected. In comparison, microbial colonies were detected in both treated and untreated AgNP/PVA scaffolds. The authors discussed the last result is ascribed to a higher dispersion diameter resulted from the AgNO 3 reduction from the thermal treatment in the PVA samples [62] (entries 8 and 16).
In the thermal reduction method, the size and spatial distribution of AgNPs can be tuned by varying the AgNO 3 solution concentration. The strategy to immobilizing and manipulating the size of the AgNPs on polymer nanofibers may be extended to other particle systems for various applications such as catalysis, energy, sensing, photonic, and biomedical applications [88]. Unfortunately, this method has been observed that leaching of AgNPs in the initial stage can occur [62] (entries 8 and 16). However, UV irradiation prepared AgNPs are preferred over those loaded on electrospun poly(vinyl alcohol) (PVA) by heat treatment of AgNO 3 for its cytotoxic and microbicidal properties.
The silver mirror reaction method produces different morphologies of Ag nanostructures (quasispheres, wires, rod, right bipyramid, beam, spheres, cube, and octahedron) which cannot be predefined [66], limiting its use for the synthesis of a particular Ag nanostructure. However, to our knowledge, none of the methods compared in this review allow us to design the nanostructure. On the other hand, the silver mirror reaction method is easily controlled, and it takes place at ambient conditions but requires further processing (filtration, purification, and washing) [64] (entry 13). Nanofiber morphology has no significant differences if prepared by SMR or the thermal reduction method [47].
Hence, from the above analysis, we can conclude that all four considered methods presented difficulties in loading AgNPs into the nanofibers, but this does not impact the antimicrobial effectivity of the nanofiber/nanoparticle systems ( Table 2). It would be interesting to compare the concentration of AgNO 3 is used and how much Ag is loaded. Still, at the moment, several studies do not evaluate the Ag loaded on the nanofibers, which does not allow to do these correlations.

Future Perspectives and Limitations
The properties as porosity, a high surface area/volume ratio, and porous interconnection that can be reached with electrospun polymeric nanofibers make them attractive in different  Journal of Nanomaterials fields [89]. Thus, in the last years, applications for filtering, tissue engineering, wound dressing, drug delivery, and biosensors have been reported using electrospun nanofibers with inorganic particles [90]. However, several variables affect the electrospinning process. Such variables are not yet fully described [48]. The electrospinning method is also limited for parameters that have to be optimized. For example, the large-scale production may present polymer blockage, and electrospinning flux may alter the electric field. It has been reported that this can be fixed using a multirow component in the injection system [91]. Also, it is desired to control nanofiber thickness with collecting devices with different characteristics [9], such as centrifugal electrospinning, or surface-free systems [92]. Other improvement points are precision and reproducibility; for those, the control of temperature and humidity is essential [9]. Finally, stability and mechanical properties can be upgraded with new electrospinning formulations [92][93][94].
The microbicidal effect of silver has been related to Ag + ion interaction with the cell membrane. It has been described that in AgNPs, Gram-negative bacteria inhibition depends on the concentration associated with cell wall damage [63], but a precise mechanism is still unknown. Still, more studies have to be implemented to investigate the mechanism of the antimicrobial action of the AgNPs. Most importantly, it is necessary to develop the ideal method that does not present any of the troubles mentioned in this review.
The literature data analysis made in the present review indicates that at the present moment, there are numerous publications dedicated to the application of different methods of deposition of AgNPs on nanofibrous scaffolds. Nevertheless, these results are not enough to conclude which method is the most promising for the best control of Ag + ion release. Research where all methods are applied in similar experimental conditions (the same silver nanoparticles type, including stabilizer, particle size, hydrodynamic diameter, and Ag concentration) is necessary to carry out to answer this question.

Conclusions
The analysis carried out in this work showed that the methods of AgNP incorporation into the electrospun nanofibers could alter the size of AgNPs, their distribution on the electrospun nanofibers, and their antimicrobial activity. The differences among the four analyzed methods also come from 11 Journal of Nanomaterials the incorporation time, method difficulty, complexity, and cost of the equipment and solvents used. The latter is not determined by the technique but by the selected nanofiber polymer. Until now, polyacrylonitrile nanofibers are the most frequently chosen system.
We found that the preferred methods are direct blending and UV irradiation. The other two methods (silver mirror reaction and thermal reduction) are less used and less studied. It was found that the UV treatment and thermal reduction methods can manipulate the size and concentration of AgNPs by varying the time of exposition. The direct blending and silver mirror reaction methods cannot control the AgNPs size, and small-sized AgNPs can be incorporated. The silver mirror reaction method's advantage is that it is a unique technique that can cover large surfaces with AgNPs.
On the other hand, taking into account the presented data, it is proposed that the best method to control the release of Ag + ions from the nanofibrous scaffolds is the SMR method because it already reported that the UV treatment and thermal reduction methods presented a leaching phenomenon after the AgNP loading on the fibers. In the case of the direct blending method, agglomerations of the AgNPs occur affecting controlled release. Moreover, the SRM method provides a homogeneous and well-defined distribution where the AgNP release can be controlled by optimizing the polymeric nanofiber's degradation.
Conjugation of well-known antimicrobial activity of AgNPs with electrospun nanofibers could take AgNP antimicrobial properties to their highest efficiency. However, up to now, the antimicrobial efficiency of AgNP-loaded nanofibers has not been explored thoughtfully. The mechanisms of antimicrobial action of AgNPs still need to be addressed. Finally, it is recommended to accompany the future performance of four methods discussed here with a comprehensive determination of physicochemical and antimicrobial characteristics of obtained AgNP-loaded nanofibers. This will allow making a more objective comparison of these methods.

Conflicts of Interest
The authors declare that there is no conflict of interest regarding the publication of this paper.