The Incorporated Drug Affects the Properties of Hydrophilic Nanofibers.

Hydrophilic nanofibers offer promising potential for the delivery of drugs with diverse characteristics. Yet, the effects of different drugs incorporated into these nanofibers on their properties remain poorly understood. In this study, we systematically explored how model drugs, namely ibuprofen, carvedilol, paracetamol, and metformin (hydrochloride), affect hydrophilic nanofibers composed of polyethylene oxide and poloxamer 188 in a 1:1 weight ratio. Our findings reveal that the drug affects the conductivity and viscosity of the polymer solution for electrospinning, leading to distinct changes in the morphology of electrospun products. Specifically, drugs with low solubility in ethanol, the chosen solvent for polymer solution preparation, led to the formation of continuous nanofibers with uniform diameters. Additionally, the lower solubility of metformin in ethanol resulted in particle appearance on the nanofiber surface. Furthermore, the incorporation of more hydrophilic drugs increased the surface hydrophilicity of nanofiber mats. However, variations in the physicochemical properties of the drugs did not affect the drug loading and drug entrapment efficiency. Our research also shows that drug properties do not notably affect the immediate release of drugs from nanofibers, highlighting the dominant role of the hydrophilic polymers used. This study emphasizes the importance of considering specific drug properties, such as solubility, hydrophilicity, and compatibility with the solvent used for electrospinning, when designing hydrophilic nanofibers for drug delivery. Such considerations are crucial for optimizing the properties of the drug delivery system, which is essential for achieving therapeutic efficacy and safety.

Influence of Polymer Concentration on Drying of SPION Dispersions by Electrospinning.

To improve the physical stability of nanoparticle dispersions, several methods for their transformation into stable and easily dispersible dry products have been investigated thus far. Recently, electrospinning was shown to be a novel nanoparticle dispersion drying method, which addresses the crucial challenges of the current drying methods. It is a relatively simple method, but it is affected by various ambient, process, and dispersion parameters, which impact the properties of the electrospun product. The aim of this study was, thus, to investigate the influence of the most important dispersion parameter, namely the total polymer concentration, on the drying method efficiency and the properties of the electrospun product. The formulation was based on a mixture of hydrophilic polymers poloxamer 188 and polyethylene oxide in the weight ratio of 1:1, which is acceptable for potential parenteral application. We showed that the total polymer concentration of prior-drying samples is closely related to their viscosity and conductivity, also affecting the morphology of the electrospun product. However, the change in morphology of the electrospun product does not affect the efficiency of SPION reconstitution from the electrospun product. Regardless of the morphology, the electrospun product is not in powder form and is therefore safer to handle compared to powder nanoformulations. The optimal total polymer concentration in the prior-drying SPION dispersion, which enables the formation of an easily dispersible electrospun product with high SPION-loading (65% (w/w)) and fibrillar morphology, was shown to be 4.2% (w/v).

The shape anisotropy of magnetic nanoparticles: an approach to cell-type selective and enhanced internalization.

The effects of the shape anisotropy of nanoparticles on cellular uptake is still poorly understood due to challenges in the synthesis of anisotropic magnetic nanoparticles of the same composition. Here, we design and synthesize spherical magnetic nanoparticles and their anisotropic assemblies, namely magnetic nanochains (length ∼800 nm). Then, nanoparticle shape anisotropy is investigated on urothelial cells in vitro. Although both shapes of nanomaterials reveal biocompatibility, we havefound significant differences in the extent of their intracellular accumulation. Contrary to spherical particles, anisotropic nanochains preferentially accumulate in cancer cells as confirmed by inductively coupled plasma (ICP) analysis, indicating that control of the nanoparticle shape geometry governs cell-type-selective intracellular uptake and accumulation.

Nanofibers with genotyped Bacillus strains exhibiting antibacterial and immunomodulatory activity.

Biofilm-associated diseases such as periodontitis are widespread and challenging to treat which calls for new strategies for their effective management. Probiotics represent a promising approach for targeted treatment of dysbiosis in biofilm and modulation of host immune response. In this interdisciplinary study, nanofibers with two autochthonous Bacillus strains 27.3.Z and 25.2.M were developed. The strains were isolated from the oral microbiota of healthy individuals, and their genomes were sequenced and screened for genes associated with antimicrobial and immunomodulatory activities, virulence factors, and transferability of resistance to antibiotics. Spores of two Bacillus strains were incorporated individually or in combination into hydrophilic poly(ethylene oxide) (PEO) and composite PEO/alginate nanofibers. The nanofiber mats were characterised by a high loading of viable spores (> 7 log CFU/mg) and they maintained viability during electrospinning and 6 months of storage at room temperature. Spores were rapidly released from PEO nanofibers, while presence of alginate in the nanofibers prolonged their release. All formulations exhibited swelling, followed by transformation of the nanofiber mat into a hydrogel and polymer erosion mediating spore release kinetics. The investigated Bacillus strains released metabolites, which were not cytotoxic to peripheral blood mononuclear cells (PBMCs) in vitro. Moreover, their metabolites exhibited antibacterial activity against two periodontopathogens, an antiproliferative effect on PBMCs, and inhibition of PBMC expression of proinflammatory cytokines. In summary, the developed nanofiber-based delivery system represents a promising therapeutic approach to combat biofilm-associated disease on two fronts, namely via modulation of the local microbiota with probiotic bacteria and host immune response with their metabolites.

Towards the development of a biorelevant in vitro method for the prediction of nanoemulsion stability on the ocular surface.

Ophthalmic oil-in-water nanoemulsions (NEs) are a complex technological platform, representing an advancement in the treatment of dry eye disease. In addition to enabling the incorporation of poorly soluble active pharmaceutical ingredients (APIs), NEs provide prolonged residence time of APIs and other formulation components and consequent replenishment and stabilization of the compromised tear film. Ophthalmic NEs have been on the market for over 20 years, but considering their complexity, as well as the complex nature of the ocular surface, they are still a poorly understood advanced dosage form. The objective of this study was to develop a biorelevant in vitro method that would be able to predict the behavior of ophthalmic NEs after application. With that goal, NE formulations differing in critical material attributes and critical formulation variables were employed and subjected to simulated tear turnover and blinking. By gradually increasing the complexity of the in vitro method, we were able to detect key parameters influencing NE stability. The undertaken study presents a step forward in the development of in vitro tools that are fundamental to the reliable, cost and time-effective development of innovative and generic topical ophthalmic NEs.

Electrospinning as a method for preparation of redispersible dry product with high content of magnetic nanoparticles.

One of the key technological challenges in the development of iron-oxide-based magnetic nanoparticles (MNPs) is their long-term physical stability in colloidal dispersions. This can be improved by their transformation into a dry form. Here, we introduce electrospinning as a drying method for ethanol-based and water-based MNP dispersions, which enables the preparation of high-loaded dry MNP products. The obtained easily dispersible electrospun product contained up to 50 % (w/w) of MNPs, homogeneously distributed in the fibrillar structure, which is much more compared to the products of currently available methods for drying MNP dispersions. The polymers used as building blocks of nanofibers, namely poloxamer 188 and polyethylene oxide, improved the tolerance of MNPs to high ionic strength dispersion medium and thus enhanced the short-term physical stability of MNP dispersions after reconstitution. The dry product was stable for up to 1 month at room temperature and relative humidity up to 70 %. It was in the form of a nanofiber mat, which prevented the aerosolization of MNPs and their unintentional ambient exposure. Therefore, the electrospun product with MNPs is expected to be a safer dry formulation of MNPs than the nanoparticulate powders, which are usually the final products of the conventional drying methods.

Hydrophilic nanofibers as a supersaturating delivery system for carvedilol.

Polymer nanofibers represent a promising delivery system for poorly water-soluble drugs; however, their supersaturating potential has not been explored yet. Here, carvedilol-loaded nanofibers based on poly(ethyleneoxide) and on amphiphilic block copolymer poloxamer 407 were produced by electrospinning. These nanofibers provided high carvedilol loading and improved dissolution of carvedilol. Their dissolution resulted in a supersaturated system that was not stable, and thus to avoid carvedilol precipitation, hydroxypropyl methylcelluloses or polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus) were additionally incorporated into the nanofibers. The morphology of the electrospun product was not affected by incorporation of carvedilol and the polymer precipitation inhibitors, as shown by scanning electron microscopy. The hydroxypropyl methylcelluloses were not effective polymer precipitation inhibitors for carvedilol. Incorporation of Soluplus significantly extended the duration of carvedilol supersaturation (>24 h) compared to the dissolution of nanofibers without Soluplus. Moreover, after 1 h of dissolution, incorporation of Soluplus into the nanofibers provided significantly higher carvedilol concentration (94.4 ± 2.5 µg/mL) compared to the nanofibers without Soluplus (32.7 ± 5.8 µg/mL), the polymer film (24.0 ± 2.2 µg/mL), and the physical mixture (3.3 ± 0.4 µg/mL). Thus, this study shows the great potential for hydrophilic nanofibers as a delivery system for sustained carvedilol supersaturation.

Treatment challenges and delivery systems in immunomodulation and probiotic therapies for periodontitis.

Introduction: Periodontitis is a widespread illness that arises due to disrupted interplay between the oral microbiota and the host immune response. In some cases, conventional therapies can provide temporary remission, although this is often followed by disease relapse. Recent studies of periodontitis pathology have promoted the development of new therapeutics to improve treatment options, together with local application using advanced drug delivery systems.Areas covered: This paper provides a critical review of the status of current treatment approaches to periodontitis, with a focus on promising immunomodulation and probiotic therapies. These are based on delivery of small molecules, peptides, proteins, DNA or RNA, and probiotics. The key findings on novel treatment strategies and formulation of advanced delivery systems, such as nanoparticles and nanofibers, are highlighted.Expert opinion: Multitarget therapy based on antimicrobial, immunomodulatory, and probiotic active ingredients incorporated into advanced delivery systems for application to the periodontal pocket can improve periodontitis treatment outcomes. Translation of such adjuvant therapy from laboratory to patient is expected in the future.

Bioevaluation methods for iron-oxide-based magnetic nanoparticles.

Despite the intensive development and unique properties of iron-oxide-based magnetic nanoparticles (MNPs), their use as drug delivery systems has not yet entered clinical practice. There also remains a lack of data on their toxicity profile and behavior in the bioenvironment. A number of in-vitro studies have been performed, but these were carried out with various MNPs using various methods of bioevaluation and various cell lines, so they are not universally applicable. It is of vital importance that selection of any experimental set-up and parameters for MNP bioevaluation, as well as the cell lines used, are focused on the final application of the MNPs. In this review, the most commonly used in-vitro methods for bioevaluation of MNPs are presented, including their key advantages and shortcomings. This critical comparison of these methods should facilitate selection of the appropriate in-vitro bioevaluation methods, and define the already established protocols that are available in the literature. Thus, we present here the first comprehensive review of in-vitro bioevaluation methods currently available for MNP evaluation. Furthermore, we provide important guidelines for selection of the best method, to enable reliable comparisons of the biological properties of different MNPs, and hence to promote their efficient translation from research to clinical practice.

The potential of nanofibers to increase solubility and dissolution rate of the poorly soluble and chemically unstable drug lovastatin.

Polymer nanofibers represent a promising drug delivery system. However, as a large surface area is exposed to external conditions during the electrospinning process, they are usually used to incorporate drugs with good oxidative stability. Here, we introduce a new concept for the incorporation of a drug with low oxidative stability and extremely low solubility into poloxamer 188/poly(ethylene oxide) and Soluplus/poly(ethylene oxide) nanofibers, to improve its solubility and increase its dissolution rate. The electrospun products showed different morphologies and lower initial lovastatin contents than theoretically expected, which indicated oxidative drug degradation during electrospinning. The addition of antioxidants improved the lovastatin chemical stability in the nanofibers. The highest lovastatin dissolution rate and solubility were obtained for the Soluplus-based nanofibers, where no crystalline lovastatin was detected. The accelerated stability study has revealed the chemical stability of lovastatin in the poloxamer-based nanofibers with the addition of antioxidants, whereas in the Soluplus-based nanofibers lovastatin was not completely preserved. However, appropriate packaging of the formulation and storage under normal conditions is expected to assure its stability. These Soluplus-based nanofibers developed here with antioxidants represent a promising immediate release formulation to provide improved solubility and dissolution rate for poorly soluble and chemically unstable drugs, such as lovastatin.

Sustained release of antimicrobials from double-layer nanofiber mats for local treatment of periodontal disease, evaluated using a new micro flow-through apparatus.

Periodontal disease is a widespread chronic condition associated with degradation of periodontal tissues that requires more effective approaches for its treatment. Thus, the aim was to develop a nanodelivery system for local application of antimicrobials, with evaluation in vitro using a newly developed micro flow-through apparatus that simulates local in-vivo conditions in the periodontal pocket: small resting volume, and low gingival crevicular fluid flow rate. We successfully developed a double-layer nanofiber mat composed of a chitosan/ poly(ethylene) oxide nanofiber layer with 30% ciprofloxacin, and a poly(ε-caprolactone) nanofiber layer with 5% metronidazole. The precisely designed composition enabled sustained in-vitro release of the antimicrobials according to their specific drug release mechanisms. The rate-limiting step of ciprofloxacin release was its own low solubility at pH 7.4, when there was excess of solid drug present in the delivery system. In contrast, sustained release of metronidazole was due to slow penetration of dissolution medium through the hydrophobic poly(ε-caprolactone) nanofiber layer. The double-layer nanofiber mat developed showed antibacterial activity against Escherichia coli and Aggregatibacter actinomycetemcomitans based on plate antibiogram assays. The antimicrobial concentrations released from the nanofiber mats determined using the developed apparatus were above the minimal inhibitory concentrations against the periodontal pathogens for up to 7 days, which is valuable information for prediction of the efficacy of the nanodelivery system. Although this apparatus was specifically designed for characterization of formulations associated with treatments for periodontal disease, its applicability is much wide, as for development of any delivery system for application at target sites that have similar local conditions.

Effects of Electrospinning on the Viability of Ten Species of Lactic Acid Bacteria in Poly(Ethylene Oxide) Nanofibers.

Lactic acid bacteria can have beneficial health effects and be used for the treatment of various diseases. However, there remains the challenge of encapsulating probiotics into delivery systems with a high viability and encapsulation efficacy. The electrospinning of bacteria is a novel and little-studied method, and further investigation of its promising potential is needed. Here, the morphology, zeta potential, hydrophobicity, average cell mass, and growth characteristics of nine different species of Lactobacillus and one of Lactococcus are characterized. The electrospinning of polymer solutions containing ~10 log colony forming units (CFU)/mL lactic acid bacteria enabled the successful incorporation of all bacterial species tested, from the smallest (0.74 µm; Lactococcus lactis) to the largest (10.82 µm; Lactobacillus delbrueckii ssp. bulgaricus), into poly(ethylene oxide) nanofibers with an average diameter of ~100 nm. All of these lactobacilli were viable after incorporation into nanofibers, with 0 to 3 log CFU/mg loss in viability, depending on the species. Viability correlated with the hydrophobicity and extreme length of lactic acid bacteria, whereas a horizonal or vertical electrospinning set-up did not have any role. Therefore, electrospinning represents a promising method for the incorporation of lactic acid bacteria into solid delivery systems, while drying the bacterial dispersion at the same time.

One-Pot Method for Preparation of Magnetic Multi-Core Nanocarriers for Drug Delivery.

The development of various magnetically-responsive nanostructures is of great importance in biomedicine. The controlled assembly of many small superparamagnetic nanocrystals into large multi-core clusters is needed for effective magnetic drug delivery. Here, we present a novel one-pot method for the preparation of multi-core clusters for drug delivery (i.e., magnetic nanocarriers). The method is based on hot homogenization of a hydrophobic phase containing a nonpolar surfactant into an aqueous phase, using ultrasonication. The solvent-free hydrophobic phase that contained tetradecan-1-ol, γ-Fe2O3 nanocrystals, orlistat, and surfactant was dispersed into a warm aqueous surfactant solution, with the formation of small droplets. Then, a pre-cooled aqueous phase was added for rapid cooling and the formation of solid magnetic nanocarriers. Two different nonpolar surfactants, polyethylene glycol dodecyl ether (B4) and our own N¹,N¹-dimethyl-N²-(tricosan-12-yl)ethane-1,2-diamine (SP11), were investigated for the preparation of MC-B4 and MC-SP11 magnetic nanocarriers, respectively. The nanocarriers formed were of spherical shape, with mean hydrodynamic sizes <160 nm, good colloidal stability, and high drug loading (7.65 wt.%). The MC-B4 nanocarriers showed prolonged drug release, while no drug release was seen for the MC-SP11 nanocarriers over the same time frame. Thus, the selection of a nonpolar surfactant for preparation of magnetic nanocarriers is crucial to enable drug release from nanocarrier.

Development of electrospun nanofibers that enable high loading and long-term viability of probiotics.

The interest in probiotics has grown in recent years due to increased awareness of the importance of microbiota for human health. We present the development of monolithic poly(ethylene oxide) and composite poly(ethylene oxide)/lyoprotectant nanofibers loaded with the probiotic Lactobacillus plantarum ATCC 8014. High loading was achieved for L. plantarum cells (up to 7.6 × 108 colony-forming unit/mg) that were either unmodified or expressing mCherry fluorescent protein. The initial concentration of L. plantarum in poly(ethylene oxide) solution was reported, for the first time, as the most critical parameter for its high viability after electrospinning, whereas the applied electric voltage and relative humidity during electrospinning did not vitally impact upon L. plantarum viability. The presence of amorphous lyoprotectant (especially trehalose) in the nanofibers promoted L. plantarum survival due to lyoprotectant interactions with L. plantarum cells. L. plantarum cells in nanofibers were stable over 24 weeks at low temperature, thereby achieving stability comparable with that in lyophilizates. The poly(ethylene oxide) nanofibers released almost all of the L. plantarum cells over 30 min, which will be adequate for their local administration. Our integrated approach enabled development of a promising nanodelivery system that provides high loading and long-term viability of L. plantarum in nanofibers, for local delivery to re-establish the microbiota balance e.g. in vagina.

Nanofibers with Incorporated Autochthonous Bacteria as Potential Probiotics for Local Treatment of Periodontal Disease.

The conventional treatment of periodontal disease does not solve the high incidence of recolonization of periodontal pockets by pathogens. Here, we introduce an innovative concept of incorporating autochthonous bacteria as potential probiotics into nanofibers for local treatment. We selected and isolated the strain 25.2.M from the oral microbiota of healthy volunteers. It was identified as Bacillus sp. based on 16S rRNA sequence analyses. The strain is nonpathogenic, produces antimicrobial substances, and can grow over the periodontal pathogen Aggregatibacter actinomycetemcomitans in vitro, making it a promising probiotic candidate. The strain 25.2.M was successfully incorporated into the nanofibers in the form of spores (107 CFU/mg), the viabilities of which were exceptional (max. change of 1 log unit) both during electrospinning and after 12 months of storage. The release of the bacteria was delayed from chitosan/poly(ethylene oxide) compared to poly(ethylene oxide) nanofibers, and the antimicrobial activity against A. actinomycetemcomitans was confirmed. The developed nanodelivery system for administration into periodontal pockets thus offers a promising approach for the inhibition of periodontal pathogens and restoration of healthy oral microbiota.

Impact of PCL nanofiber mat structural properties on hydrophilic drug release and antibacterial activity on periodontal pathogens.

Electrospinning enables to design and manufacture novel drug delivery systems capable of advancing the local antibacterial therapy. In this study, two hydrophilic drugs - metronidazole and ciprofloxacin hydrochloride - were loaded both individually and in combination into hydrophobic poly(ε-caprolactone) (PCL) matrix using electrospinning. We aimed to develop prolonged release drug delivery systems suitable for the treatment of periodontal diseases and understand how different rarely studied structural features, such as nanofiber mat thickness, surface area, wettability, together with intrinsic properties, like solid state and localization of incorporated drugs in nanofibers, affect the drug release. Furthermore, the safety of nanofiber mats was assessed in vitro on fibroblasts, and their antibacterial activity was tested on selected strains of periodontopathogenic bacteria. The results showed that the structural properties of nanofiber mat are crucial in particular drug-polymer combinations, affecting the drug release and consequently the antibacterial activity. The hydrophobicity of a PCL nanofiber mat and its thickness are the key characteristics in prolonged hydrophilic drug release, but only when wetting is the rate-limiting step for the drug release. Combination of drugs showed beneficial effects by inhibiting the growth of all tested pathogenic bacterial strains important in periodontal diseases.

Association of Dyslipidemia, Oxidative Stress, and Inflammation With Redox Status in VLDL, LDL, and HDL Lipoproteins in Patients With Renal Disease.

Some cardiovascular complications in patients with chronic kidney disease and end-stage renal disease may be caused by structurally and functionally modified lipoproteins. Redox status (advanced oxidation protein products [AOPPs]), prooxidant-antioxidant balance, total protein sulfhydryl (SH-groups), and paraoxonase 1 (PON1) activity were assessed in 77 renal patients and 20 controls. Lipoproteins were isolated using ultracentrifugation. PON1, PON3, and pentraxin-3 concentration were determined by enzyme-linked immunosorbent assay (ELISA). Dyslipidemia-Oxy-Inflammation (DOI) score was calculated as a sum of dyslipidemia, oxidative stress, and inflammation scores. The dyslipidemia score ( P < .001), oxy score ( P < .01), inflammation score (P < .001), and the DOI score ( P < .001) were higher in patient groups compared with controls. The very-low-density lipoprotein (VLDL) fraction contained the highest amount of AOPP ( P < .001) compared with other lipoprotein fractions in all groups. The low-density lipoprotein (LDL) fraction contained elevated AOPP in all groups compared with the high-density lipoprotein (HDL) fraction ( P < .001). Significant positive correlation was observed between AOPP in LDL fraction and DOI score (ρ = 0.510, P < .01). Dyslipidemia, oxidative stress, and inflammation play an interactive role in renal disease and are mutually associated with redox status in VLDL, LDL, and HDL lipoproteins in plasma of renal patients.

Preparation of poloxamer-based nanofibers for enhanced dissolution of carvedilol.

Polymer nanofibers have become increasingly important for improvement of dissolution and bioavailability of poorly soluble drugs, representing a great challenge in pharmaceutical development. Here, we introduced a new concept of using amphiphilic polymers as fundamental excipients in electrospun nanofibers, which would improve drug solubilization and accelerate its release. Hydrophilic poloxamer-based nanofibers were developed as a novel drug delivery system for carvedilol. These nanofibers were electrospun from different liquid carvedilol dispersions, as carvedilol (nano)suspensions or ethanol solution. The electrospun products showed similar morphologies, but different mean fiber diameters (170-450 nm). Carvedilol dissolution rates from nanofibers were faster compared to its dissolution from polymer films. The electrospinning from ethanol solution resulted in the highest dissolution rate, since >90% of the drug was dissolved in the first 5 min. The type of liquid medium significantly affects also the drug crystallinity. Thus, nanofibers produced from ethanol polymer solution showed no detectable crystalline carvedilol, whereas crystalline carvedilol form II or III was detected in the other nanofiber samples investigated. In a prolonged stability study (to 1 year), the potential of nanofibers to preserve the active ingredient in the initial non-crystalline form was demonstrated. Poloxamer-based nanofibers thus represent a promising formulation for immediate release and improved dissolution rates of poorly soluble drugs.

Design and Fabrication of Magnetically Responsive Nanocarriers for Drug Delivery.

Magnetically-assisted delivery of therapeutic agents to the site of interest, which is referred to as magnetic drug targeting, has proven to be a promising strategy in a number of studies. One of the key advantages over other targeting strategies is the possibility to control remotely the distribution and accumulation of the nanocarriers after parenteral administration. However, preparation of effective and robust magnetically responsive nanocarriers based on superparamagnetic iron oxide nanocrystals (SPIONs) still represents a great scientific challenge, since spatial guidance of individual SPIONs is ineffective despite the presence of high magnetic field gradient. A strategy to overcome this issue is the clustering of SPIONs to achieve sufficient magnetic responsiveness. In this mini-review, we address current and future strategies for the design and fabrication of magnetically responsive nanocarriers based on SPIONs for magnetically-targeted drug delivery, including the underlying physical requirements, the possibility of drug loading, and the control of drug release at the targeted site.

Formulation and evaluation of chitosan/polyethylene oxide nanofibers loaded with metronidazole for local infections.

Nanofibers combined with an antimicrobial represent a powerful strategy for treatment of various infections. Local infections usually have a low fluid volume available for drug release, whereas pharmacopoeian dissolution tests include a much larger receptor volume. Therefore, the development of novel drug-release methods that more closely resemble the in-vivo conditions is necessary. We first developed novel biocompatible and biodegradable chitosan/polyethylene oxide nanofibers using environmentally friendly electrospinning of aqueous polymer solutions, with the inclusion of the antimicrobial metronidazole. Here, the focus is on the characterization of these nanofibers, which have high potential for bioadhesion and retention at the site of application. These can be used where prolonged retention of the delivery system at an infected target site is needed. Drug release was studied using three in-vitro methods: a dissolution apparatus (Apparatus 1 of the European Pharmacopoeia), vials, and a Franz diffusion cell. In contrast to other studies, here the Franz diffusion cell method was modified to introduce a small volume of medium with the nanofibers in the donor compartment, where the nanofibers swelled, eroded, and released the metronidazole, which then diffused into the receptor compartment. This set-up with nanofibers in a limited amount of medium released the drug more slowly compared to the other two in-vitro methods that included larger volumes of medium. These findings show that drug release from nanofibers strongly depends on the release method used. Therefore, in-vitro test methods should closely resemble the in-vivo conditions for more accurate prediction of drug release at a therapeutic site.