Loading and Release of Quercetin from Contact-Drawn Polyvinyl Alcohol Fiber Scaffolds

Abstract

Polymeric drug releasing systems have numerous applications for the treatment of chronic diseases and traumatic injuries. In this study, a simple, cost-effective, and scalable method for dry spinning of crosslinked polyvinyl alcohol (PVA) fibers is presented. This method utilizes an entangled solution of PVA to form liquid bridges that are drawn into rapidly drying fibers through extensional flow. The fibers are crosslinked by a one-pot reaction in which glyoxal is introduced to the PVA solution prior to contact drawing. Failure analysis of fiber formation is used to understand the interplay of polymer concentration, glyoxal concentration, and crosslinking time to identify appropriate formulations for the production of glyoxal-crosslinked PVA fibers. The small molecule quercetin (an anti-inflammatory plant flavonoid) can be added to the one-pot reaction and is shown to be incorporated into the fibers in a concentration-dependent manner. Upon rehydration in an aqueous medium, the glyoxal-crosslinked PVA fiber scaffolds retain their morphology and slowly degrade, as measured over the course of 10 days. As the scaffolds degrade, they release the loaded quercetin, reaching a cumulative release of 56 ± 6% of the loaded drug after 10 days. The bioactivity of the released quercetin is verified by combining quercetin-loaded fibers with contact-drawn polyethylene oxide-type I collagen (PEO-Col) fibers and monitoring the growth of PC12 cells on the fibers. PC12 cells readily attach to the PEO-Col fibers and display increased nerve growth factor-induced elongation and neurite formation in the presence of quercetin-loaded PVA fibers relative to substrates formed from only PEO-Col fibers or PEO-Col and PVA fibers without quercetin.

Local administration of anti-inflammatory compounds has received considerable attention for treatment of traumatic injuries to soft tissues (e.g., nerve tissue), while minimizing complications of systemic administration such as off-target toxicity or side effects, fluctuations in drug concentrations above and below therapeutic thresholds, and issues associated with stability of drugs with short half-lives.^1,2 As such, a variety of natural and synthetic materials have been explored for their suitability to release drugs at sites of injury.^3,4 Synthetic materials for drug release can be fabricated by techniques including (but not limited to) electrospinning, photo/soft-lithography, 3D-printing, solvent casting, freeze-drying, and hydrogel formation.⁵⁻⁸ Well defined physicochemical characteristics, as is typically the case for synthetic materials, enable a wide range of chemical modifications useful for tuning the mechanical properties and degradation profile of a material.⁹ The most common synthetic materials for drug release include glycolic acid derivatives, lactic acid derivatives, and polyester derivatives, all of which can be processed to provide scaffolds with form factors that enable their delivery to a site of injury (e.g., particles that can be injected, or foams, gels, or textiles that can be placed at the site of injury or at a nearby site within the tissue).¹⁰

Small molecule therapeutics, viral vectors and other forms of gene transfer agents, and protein-based adjuvants such as growth factors and monoclonal antibodies display improved efficacy when delivered locally from a scaffold compared to when they are taken orally or delivered by intravenous injection.¹¹⁻¹⁴ In the context of peripheral nerve repair, injectable hydrogels have served as scaffolds for drug release.^15,16 Photopolymerized hydrogel scaffolds formed from polyethylene glycol (PEG) and PEG-polylactic acid (PLA) derivatives have been shown to release neurotrophin-3 over a period of 50 days, promoting axonal sprouting and improving functional recovery in rats following spinal cord injury.^17,18 Natural scaffold materials formed from collagen fibers have also shown utility for loading and release of neurotrophin-3 to improve functional recovery following spinal cord injury in rats.¹⁹ In addition, synthetic derivatives of natural materials (e.g., chitosan) have been used to promote nerve regeneration and functional recovery following nerve injury in rats by way of local delivery of anti-inflammatory compounds such as curcumin.²⁰ Delivery of curcumin was also shown to improve peripheral nerve regeneration when loaded into a silicone conduit.²¹

Ultimately, the performance of the scaffold depends not only on the types and amounts of adjuvants that can be loaded and released but also on the form factor of the material, with fibrous materials offering numerous advantages with respect to the surface area to volume ratio, control of the scaffold porosity, ease of handling, postprocessing, and higher-order assembly, as well as ease of integration as a component of a composite material.²² Fibrous scaffolds are typically produced by wet spinning, melt spinning, direct spinning, extrusion spinning, or electrospinning.^23,24 Of these techniques for generating fibers, electrospinning offers distinct advantages in terms of throughput and the ability to precisely tune the fiber diameter from ∼2 nm up to several microns. Many types of electrospun scaffolds have been proposed for nerve repair, including those formed from hemin-doped serum albumin, polyvinylidene fluoride, polycaprolactone–gelatin, and polyvinyl alcohol (PVA).²⁵⁻²⁸ As a notable example and a basis of comparison to the present study, PVA–gelatin–gellan was loaded with quercetin prior to electrospinning to produce mats of PVA fibers, which were then rolled into circular conduits.²⁹ Other notable examples of small molecule drug release from PVA fibers include pyrroloquinoline quinone (an antioxidant and anti-inflammatory agent with neuroprotective effects) and phycocyanin (an anti-inflammatory, neuroprotective, and antioxidant compound).^30,31

Electrospinning is not without its disadvantages, however, which include relatively small pore sizes in electrospun scaffolds due to ultrafine fiber diameters, close spacing between fibers, the use of toxic and volatile solvents to ensure homogeneity of the spin dope and aid in the drying of the fibers, shear stresses and electrical fields that may damage labile compounds, and issues associated with nozzle clogging and jet instability, which make it difficult to scale production of electrospun scaffolds.³²⁻³⁴ In the case of water-soluble polymers such as PVA, it is also necessary to perform crosslinking after the fibers are deposited.^32,35 To address some of the limitations of electrospinning, nozzle-free contact drawing techniques have been developed. Contact drawing works by dissolving a high molecular weight polymer in water above the critical concentration at which polymer entanglement occurs, interposing the entangled polymer solution between two substates, which are pulled apart to first generate a series of liquid bridges, and then drawing the polymer solution through extensional flow into a growing fiber that dries rapidly in air as it elongates.³⁶ This approach has been used previously with dextran as the high molecular weight polymer to load antibiotics, hemostatic agents, and collagen into fibers,^37,38 and with polyethylene oxide to load collagen and silver nanoparticles into fibers.^39,40

Here, we applied the contact drawing approach to generate PVA fibers for the release of quercetin, a plant flavonoid with anti-inflammatory and neuroprotective effects.^41,42 Quercetin is best known for its antioxidative^43,44 and antimicrobial properties.⁴⁵ Quercetin has been shown to interact with various signaling pathways, for example, in suppression of tissue necrosis factor-α induced apoptosis and inflammation,⁴⁶ modulation of sirtuin proteins,⁴⁷ nuclear translocation of nuclear factor erythroid 2-related factor 2 to induce expression of enzymes involved in antioxidant synthesis,⁴⁸ and inhibition of c-Jun N-terminal kinases/mitogen-activated protein kinase.⁴⁹ A simple one-pot crosslinking reaction with glyoxal was carried out as the fibers were formed. Assembling the fibers into a nonwoven scaffold enabled the release of quercetin over the course of 10 days in artificial cerebrospinal fluid (aCSF). The scaffold swelling and degradation profiles were consistent with the measured quercetin release profile. When combined with contact-drawn polyethylene oxide-type I collagen (PEO-Col) fibers, the quercetin-loaded fibers enhanced nerve growth factor (NGF)-induced cell elongation and neurite formation in PC12 cells, thus supporting potential applications in peripheral nerve regeneration.

Experimental Section

Contact Drawing of PVA Fibers

PVA (Mowiol 40–88, 205 kDa; Sigma-Aldrich) was dissolved in distilled water. For generating single fibers, a microneedle and a 3D printed reservoir were mounted on a linear translation stage (Thorlabs) (Figure S1).³⁶ Approximately 50 μL of PVA solution was added to the reservoir using a disposable transfer pipette. To form a single fiber, the needle first penetrated the surface of the PVA solution to a depth of ∼2–3 mm. After a 3 s delay, the needle was withdrawn from the solution at a set speed (40–400 mm/s) over a total path length of 100 mm. For generating the fiber scaffolds, a 3D-printed pin array tool comprised of cylindrical pins with flat caps (height 5 mm, radius 0.75 mm, and edge-to-edge spacing 0.5 mm) was used. The pin array tool was manually brought into contact with a flat substrate tool coated with the polymer solution (<1 mm penetration depth) and was then pulled away to generate fibers, thus constituting a single fiber elongation cycle. The fibers were then deposited either onto glass slides for microscopic analysis or over an empty beaker for use in scaffold degradation and quercetin release studies (Figure 1A). The reservoir, pin array, and substrate tool were fabricated using a B9 Creator 3D printer using B9R-2 Black resin (B9 Creations). Detailed designs for the reservoir, pin array tool, and substrate tool have been reported previously.^36,40

Figure 1.

Contact drawing of glyoxal-crosslinked fibers. (A) Contact drawing process begins with the preparation of a fiber-forming solution at a concentration that exceeds the entanglement concentration for a specified molecular weight of the polymer (in this case, 205 kDa PVA). To this viscous polymer solution, glyoxal can be added to initiate a one-pot crosslinking reaction. Fibers can then be formed by a three-step process in which: (1) PVA/glyoxal solution is applied to a flat substrate; (2) thin layer of PVA on the substrate is contacted with a nucleating element, such as a single needle or an array of 3D-printed pins; and (3) nucleating element and substrate tool are pulled apart to elongate fibers that rapidly dry under ambient conditions. A glyoxal vapor treatment can be applied to further stabilize the dry fibers. (B) Reaction scheme for glyoxal-mediated crosslinking of PVA. (C) Images of the dry fibers crosslinked by the one-pot addition of glyoxal, glyoxal vapor crosslinking after contact drawing, and combined one-pot and vapor crosslinking. Scale bars are 100 μm.

Failure Analysis to Identify Favorable Conditions for Fiber Formation

Fibers were pulled at various speeds corresponding to different τ_pull values,³⁶ as calculated according to

1

A total of 10 technical replicates were performed for each formulation and pull speed, until conditions were identified where failure occurred 0 and 100% of the time. A failure was recorded when a stable liquid bridge failed to form or when the fiber detached from either the reservoir or the needle before the stage returned to its set point. Failure rate was calculated using

PVA concentrations of 7.5, 10.0, 12.5, 15.0, and 17.5 wt % were examined. Mol effective glyoxal concentrations of 5, 10, 20, 50, and 100% were examined for a fixed PVA concentration of 12.5 wt % 15 min after glyoxal addition. Finally, the time following glyoxal addition was examined at 5 min intervals between 15 and 45 min for a fixed PVA concentration of 12.5 wt % and a fixed glyoxal concentration of 20% mol effective. The fluid in the reservoir was replaced whenever the pull speed was adjusted (10 pulls took ∼1 min to perform). Failure rate data were fitted with a Weibull growth distribution function using Minitab.

Preparation of Crosslinked PVA Fibers and Fiber Scaffolds

One-pot crosslinking, vapor crosslinking, and a combination of one-pot and vapor crosslinking were carried out using glyoxal to form acetal linkages at free hydroxyl groups⁵⁰ (Figure 1B). For one-pot crosslinking, glyoxal was added to the PVA solution at a 20% mol effective concentration. The solution pH was lowered to 4.0 through dropwise HCl addition, and then, it was allowed to react for 15 min at room temperature. Fibers were then contact-drawn and allowed to air dry in a fume hood overnight. For vapor crosslinking, fibers were placed in sealed 473 mL chambers containing 50 mL of 40 wt % glyoxal. The chambers were heated to 60 °C in an oil bath for 12 h. The fibers were then air dried in a fume hood overnight. Quercetin-loaded fiber scaffolds were crosslinked using only the one-pot method. Fibers were pulled across glass microscopy slides for either direct analysis of one-pot crosslinked fibers or subsequent vapor crosslinking. The fibers were visualized using a Nikon Eclipse Ti optical microscope equipped with a 20× magnification objective lens. Images were collected prior to rehydration in distilled water, and at 3, 12, and 48 h after rehydration.

Infrared Spectroscopy of PVA Films

Fourier transform infrared (FTIR) spectroscopy (Nicolet iZ10; Thermo Scientific) was used to evaluate the glyoxal crosslinking process. PVA was dissolved in distilled water at 60 °C until a clear, homogenous solution formed. To this solution, glyoxal was added to achieve concentrations of 0, 20, 40, 60, and 100% mol effective. The pH was then lowered to 4.0 through the dropwise addition of HCl, and the reaction was allowed to proceed at 60 °C for 2 h. The resulting viscous material was then poured onto a flat polydimethylsiloxane (PDMS; Sylgard 184; Dow Corning) substrate and air dried overnight in a fume hood to form films. The resulting films were dried in a gravity convection oven at 40 °C for an additional 2 h prior to FTIR spectroscopy and then directly placed on the crystal surface of a Smart iTX Sampling Accessory equipped with an AR Diamond Crystal Plate (Themo Scientific). FTIR spectra were acquired for each film using attenuated total reflection (ATR) with a total of 16 scans collected using a KBr beam splitter with a resolution of 4.00. Transmission over the wavenumber range of 400–4000 cm^–1 was evaluated, and the OMNIC software suite was used to process and view the spectral data, with background correction for the ATR crystal in the absence of a sample. The advanced ATR correction algorithm within the OMNIC software was used to process spectra to correct data points against a library of transmission spectra for relative shifts in band intensity and absolute shifts in frequency introduced by the instrument configuration.

Analysis of Quercetin Fluorescence in PVA Fibers

Quercetin was dissolved in dimethyl sulfoxide (DMSO) and added at a final concentration of 0, 50, 100, or 150 μM to the PVA/glyoxal solution. The amount of quercetin loaded into fibers was monitored by fluorescence microscopy using a series of single fibers drawn across glass microscope slides. The slides were then immersed in distilled water and immediately inspected using a Nikon Eclipse Ti optical microscope equipped with a 20× objective lens. Epifluorescence microscopy images were collected for each loading condition under identical acquisition settings. Micrographs were processed in ImageJ to correct for fiber density (i.e., number of fibers per viewing field), background fluorescence, and autofluorescence to compare fluorescence among quercetin loading conditions (Figure S2). The relative levels of quercetin present in the fibers were expressed as percent fluorescence according to

3

Corrected values were expressed by subtracting the % fluorescence of fibers containing 0 μM quercetin from the % fluorescence of fibers containing various concentrations of quercetin to account for autofluorescence of the PVA fibers.

Analysis of Scaffold Degradation and Quercetin Release

Scaffolds formed from PVA fibers were incubated in aCSF (119 mM NaCl, 26.2 mM NaHCO₃, 2.5 mM KCl, 1 mM NaH₂PO₄, 1.3 mM MgCl₂, and 10 mM glucose in distilled water⁵¹) to assess quercetin release, swelling, degradation, and structural changes. Scaffolds formed from PVA fibers without or with loading of 200 μM quercetin were prepared for analysis as described in Failure Analysis to Identify Favorable Conditions for Fiber Formation. Scaffolds were incubated in 10 mM glycine buffer to quench free aldehyde groups introduced during the crosslinking process and then dried in a gravity convection oven at 40 °C for 2 h before initial dry weight measurements were recorded. Each scaffold (38.9–61.7 mg dry weight) was then placed in 10 mL of aCSF and incubated at 37 °C for 10 days. At various time points, samples were removed from the aCSF, rinsed with distilled water, and dried at 40 °C until weight measurements were stable. The final dry weights were then recorded, and the percentage of the scaffold remaining was calculated according to

4

At 1 h, 1 day, 3 days, and 10 days, scaffolds incubated in aCSF were prepared for scanning electron microscopy (SEM), with a scalpel used to prepare cross-sectional segments. The samples were affixed to adhesive carbon tape on SEM stubs and sputter coated with gold/palladium (80/20) using a LEICA EM 600 high vacuum sputter coater at a current of 30 mA and a sputter rate of 0.03 nm/s until a coating depth of 12.19 nm was reached. SEM analysis was performed using a ZEISS Sigma 300 field emission scanning electron microscope at an accelerating voltage of 5.0 kV. Micrographs were acquired using the secondary electron detector.

To assess swelling, fibers were drawn across microscope slides as described above. Fibers were air dried in a fume hood overnight and then incubated with a 10 mM glycine solution before being dried again for 2 h in a 40 °C dry gravity convection oven. The fibers were then immersed in aCSF at 37 °C. Fibers were visualized using a Nikon Eclipse Ti optical microscope equipped with a 20× objective lens over the course of 7 days. A total of 25 fibers from each condition and each time point were analyzed in ImageJ to obtain average fiber diameters that could be used to calculate the fiber swelling over time according to

5

Quercetin release in aCSF was measured over the course of 10 days by UV/vis spectroscopy. Absorbance at 375 nm was measured using an Olis 8452A diode array spectrophotometer. The concentration of quercetin in the aCSF was interpolated from a standard curve of known quercetin concentrations versus absorbance at 375 nm (Figure S3).

Preparation of PEO-Col/PVA Fiber Substrates for PC12 Cell Culture

PC12 cells were cultured on PEO (1.5 wt %)-collagen (0.6 wt %) and PVA (12 wt %) fibers immobilized on PDMS. To prepare the PEO-Col fibers, PEO powder (8 MDa; Sigma-Aldrich) was dissolved in type I collagen solution (6 mg/mL in 0.01 M HCl, Collagen Solutions). The PEO-Col solution was prepared as described previously.³⁹ PVA fibers were prepared as described above. The elastomeric support was also prepared as described previously.³⁹ To prepare substrates with only PEO-Col fibers, a total of 30 successive elongation cycles were performed. To prepare substrates with both PEO-Col and PVA fibers present, 15 successive elongation cycles were performed for each type of solution (e.g., 15 cycles for PEO-Col followed by 15 cycles for PVA). After each elongation cycle, the fibers were lowered to contact and adhere to the surface of the PDMS. The process was performed at 20 °C and ≤40% relative humidity. All samples were then dried overnight. The elastomeric sheets with fibers were cut into ∼1 × 1 cm square pieces, which were then mounted within 24-well plates using PDMS to glue them in place.

PC12 Cell Culture

PC12 cells (ATCC CRL 1721.1) were cultured in RPMI 1640 medium (Corning), supplemented with 10% horse serum, 5% fetal bovine serum, and 1% antibiotic–antimycotic solution (Corning) in a humidified incubator at 37 °C with 5% CO₂. All cell culture substrates were UV sterilized for 30 s at 200 mJ/cm². PEO-collagen fibers were hydrated with Dulbecco’s phosphate-buffered saline (PBS; VWR) for 30 min. PEO-Col/PVA fibers were washed with glycine (10 mM in PBS) for 30 min followed by a 5 min wash with distilled water. PC12 cells were seeded at 5 × 10⁴ cells/well. After 3 days of culture, the medium was supplemented with NGF-β (100 ng/mL; Sigma-Aldrich). The PC12 cells were maintained in a medium containing NGF for an additional 7 days (replenished once after 72 h). PC12 cell morphology was observed under brightfield illumination using the Nikon Eclipse Ti optical microscope. After 10 days, the cell culture medium was removed and replaced with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT; 5 mg/mL; Sigma-Aldrich) solution. After 4 h of incubation at 37 °C, the resulting formazan crystals were dissolved in 1 mL of DMSO. The optical density was measured at 570 nm using a FilterMax F5 plate reader (Molecular Devices).

Results and Discussion

Contact drawing provides a potential route for large-scale production of biomaterial fibers from polymer solutions without the need for elaborate instrumentation, and with minimal process control requirements. The key process control considerations for contact drawing are the formulation of the fiber precursor solution and the speed at which the fibers are drawn from the precursor solution.^36,52 Contact drawing also enables blending of a wide array of potential adjuvants into the polymer precursor solution prior to contact drawing, as demonstrated here with quercetin and type I collagen. In addition, it allows one fiber precursor solution to be exchanged for another to fabricate multicomponent fiber scaffolds with a delay of only a few seconds to switch between sets of pins and substrate tools. Contact-drawn fibers dry within 1–2 s of forming, allowing the fibers to be rapidly assembled into nonwoven textiles through consecutive fiber elongation cycles.^38,40 The number of fibers produced in each elongation cycle scales with the number of pins present on the pin array tool, and the angular orientation of fibers between layers can be controlled by rotating the substrate on which the fibers are collected. In the case of PEO-Col fibers, the collagen self-assembles into a stable protein fiber upon dissolution of the PEO.³⁹ However, as PVA is water soluble,⁵³ it is necessary to determine an optimal crosslinking strategy to stabilize contact-drawn PVA fibers in aqueous environments. To ensure homogenous crosslinking of the fibers, we developed a one-pot crosslinking reaction compatible with the contact drawing process.

Glyoxal Crosslinking of PVA Fibers

Both the one-pot and glyoxal vapor crosslinking methods were postulated to allow the formation of acetal linkages to crosslink PVA (Figure 1B), thus rendering the PVA fibers less susceptible to hydrolysis and dissolution. In comparing these methods, vapor crosslinking alone led to PVA fibers that completely dissolved in distilled water after 48 h. The one-pot method alone and the combination of one-pot and vapor crosslinking methods, conversely, produced fibers that persisted after 48 h. This enhanced resistance to hydrolysis and dissolution seems likely to be due to homogenous crosslinking throughout the polymer matrix, as opposed to crosslinking of only the parts of the material that are accessible to the glyoxal vapor. As the combined one-pot and vapor crosslinking methods did not produce appreciably better fibers in terms of fiber formation rate and resistance to hydrolysis than the one-pot method alone (Figure 1C), the one-pot method was selected for further evaluation of the crosslinking reaction and its efficiency by FTIR spectroscopy.

FTIR spectra acquired for films of glyoxal-crosslinked PVA were consistent with acetal formation (Figure 2). Compellingly, some FTIR features were modulated in a glyoxal concentration-dependent manner, while others were not. As the glyoxal concentration was increased, the presence of a broad band consistent with hydroxyl stretching⁴³ from 3000 to 3500 cm^–1 decreased in intensity. This decrease in intensity is indicative of the depletion of hydroxyl groups that would initially be present in PVA, which is consistent with the formation of acetal linkages between the PVA chains.

Figure 2.

IR spectra for glyoxal-crosslinked PVA. IR spectra were collected for PVA films crosslinked through a one-pot reaction with 0, 20, 40, 60, and 100% mol effective concentrations of glyoxal. An increase in transmittance is observed between 3000 and 3500 cm^–1, indicating a decrease in O–H stretching as acetal linkages are formed between hydroxyl groups on the PVA with the increasing concentration of glyoxal.

Optimization of the Contact Drawing Process for PVA Fibers

Contact drawing relies on the formation of a liquid bridge that becomes elongated into a rapidly drying filament through extensional flow.^36,52 The formation of the liquid bridge is driven by the process of polymer entanglement.⁵⁴ For a polymer solution in the entangled regime, there is a characteristic time scale above which the polymer molecules can escape entanglement and move freely with respect to each other.^53,55 Thus, an increase in either the polymer concentration or the molecular weight of the polymer is expected to lead to increased entanglement along with fiber formation at slower pull speeds corresponding to longer τ_pull values.³⁶

As expected, increasing the concentration of PVA allowed fibers to form at slower pull speeds (Figure 3A). From these data, an optimal PVA concentration can be selected corresponding to pull times that can be easily achieved either through automated or manual contact drawing processes. Here, 12.5 wt % PVA was selected for further investigation because it reliably formed fibers both by automated single pin drawing and by hand drawing with a multipin array tool. Crosslinking PVA with glyoxal increased the effective molecular weight of the already entangled polymer solution and, therefore, allowed fiber formation to occur at slower pull speeds. Figure 3B shows the characteristic shift of the Weibull distribution toward longer pull times for a 12.5 wt % solution of PVA as a function of increasing glyoxal concentration. From these data, a 20% mol effective concentration of glyoxal was selected to examine crosslinking time, as this was the minimum crosslinking concentration that displayed a pronounced shift in the Weibull distribution.

Figure 3.

Fiber formation as a function of pull time for various formulation chemistries. (A) Failure rate versus pull time for increasing concentration of PVA. (B) Failure rate versus pull time for increasing concentration of glyoxal. (C) Failure rate versus pull time for increasing the glyoxal crosslinking time from 15 to 30 min. (D) Failure rate versus pull time for increasing the glyoxal crosslinking time from 30 to 45 min. Inset schematics depict models for entanglement of PVA chains (blue strands) and crosslinking (orange segments). Lines show Weibull growth distribution functions fit to each set of data (R² > 0.95) in each case.

For a solution of 12.5 wt % PVA with 20% mol effective glyoxal, the Weibull distribution shifted toward longer pull times with increasing crosslinking time (Figure 3C). This is consistent with the formation of additional covalent crosslinks over time and a reaction time-dependent increase in effective chain lengths. However, this behavior transitioned after 30 min of crosslinking, with the Weibull distribution shifting back toward shorter pull times (Figure 3D). After 30 min, it is possible that the polymer system has begun to form a gel. If gelation occurs, the probability of removing a polymer chain from the entangled system becomes much lower, and therefore, fibers only reliably form at short pull times. After 30 min, it is also possible that the solution in the reservoir may be heterogeneous, with regions of varying viscosity that influence the ability to form position-dependent fibers. If this were the case, the fibers observed would likely originate from regions with lower relative viscosity, as high-viscosity gel-like regions would have too many crosslinks for polymer molecules to move freely into the elongating fibers. Fiber diameter did not vary with pull speed (Figure S4). However, for a fixed pull speed, 12.5 wt % PVA fibers increased in diameter with glyoxal concentration from 2.18 ± 0.18 μm for 5% mol effective glyoxal to 3.83 ± 0.51 μm for 20% mol effective glyoxal (Figure S5). The increase in the fiber diameter with the addition of glyoxal may be explained by a reduction in the secondary flow of fluid back to the liquid reservoir and the needle tip with increased crosslinking of the polymer, thereby retaining more of the polymer solution in the elongating fiber. A similar effect was observed previously with dextran solutions that varied by molecular weight and concentration.³⁶

Loading of Quercetin into PVA Fibers

In nervous system tissue, quercetin has shown promise in limiting apoptotic cell death, fibrosis, and scar formation after injury, supporting its use as a model drug in the context of nerve repair and regeneration.^41,49 It has also been reported that quercetin induces neuronal differentiation and potentiates the effect of NGF in cultured PC12 cells.⁵⁷⁻⁵⁹ Quercetin is fluorescent, enabling the quantification of its incorporation into a drug-releasing scaffold by fluorescence microscopy.⁶⁰ The fluorescence intensity of crosslinked PVA fibers increased linearly with the concentration of quercetin added to the one-pot reaction (Figure 4). Since the contact-drawn PVA fibers are faintly autofluorescent when excited with blue light, the observed fluorescence intensities were corrected by subtracting the background fluorescence measured from fibers without quercetin (Figure 4B), enabling nondestructive quality control of the quercetin loading process. The addition of quercetin increased the fiber diameter, with more pronounced effects at lower glyoxal concentrations (Figure S5), suggesting that quercetin may participate in the crosslinking reaction to some extent. With promising evidence of quercetin incorporation and a fiber formulation and crosslinking protocol that prevents rapid degradation and solubility, bulk scaffolds were prepared for in vitro studies.

Figure 4.

Evaluation of quercetin loading in PVA fibers by fluorescence microscopy. (A) Brightfield and fluorescence microscopy images of rehydrated glyoxal-crosslinked PVA fibers loaded with various concentrations of quercetin. Scale bars are 50 μm. (B) Quantification of relative (black) and corrected (orange) fluorescence for fibers loaded with various concentrations of quercetin. A linear function provided a good fit to each data series (R² > 0.99). Error bars represent the standard error of the mean (n = 25).

PVA Scaffold Degradation in aCSF

Degradation profiles indicated a relatively stable material irrespective of quercetin loading (Figure 5D). The scaffold containing fibers loaded with quercetin degraded more rapidly than the scaffold containing fibers without quercetin at time points up to 1 day in aCSF, whereas between 1 and 3 days of incubation in aCSF the degradation profiles with and without quercetin in the fibers were indistinguishable. At time points greater than 3 days, however, the scaffold containing fibers loaded with quercetin degraded less than that without quercetin. After 10 days in aCSF, the scaffolds formed from fibers containing quercetin retained 72.5 ± 2.5% of their dry weight, whereas scaffolds formed from fibers without quercetin retained 67.1 ± 5.3% of their dry weight.

Figure 5.

Degradation of glyoxal-crosslinked PVA fiber scaffolds. (A) SEM images showing the surface morphology of glyoxal-crosslinked PVA fibers with and without loaded quercetin over the course of 10 days of incubation in aCSF. Scale bars are 50 μm. (B) SEM images showing the cross-sectional morphology of glyoxal-crosslinked PVA fibers with and without loaded quercetin over the course of 10 days of incubation in aCSF. Scale bars are 50 μm. (C) Swelling over time in aCSF for glyoxal-crosslinked PVA fibers with (black squares) and without (grey circles) loaded quercetin. (D) Scaffold degradation over time in aCSF for glyoxal-crosslinked PVA fibers with (black squares) and without (grey circles) loaded quercetin. The concentration of quercetin in the one-pot reaction was 200 μM. Error bars represent the standard error of the mean (n = 3).

The contact-drawn PVA fibers detailed here persist longer than previously reported electrospun PVA fibers crosslinked in a solution of 25% wt/vol glutaraldehyde.²⁸ Namely, by 10 days, the electrospun materials were almost entirely degraded in PBS,²⁸ whereas more than two-thirds of the dry weight of contact-drawn glyoxal-crosslinked PVA fibers remained after 10 days in aCSF. Providing additional versatility, in the case of contact-drawn PVA fibers, the degree of crosslinking can be modified within the range of conditions identified in Figure 3, potentially allowing the degradation rate to be tuned to achieve a desired profile. As shown in Figure 1, the fibers produced through this one-pot crosslinking reaction can also be further crosslinked with glyoxal vapor, providing the option to modulate the susceptibility to degradation through surface crosslinking or, potentially, through immersion in a crosslinking solution after fiber formation.

PVA fibers without quercetin decreased in diameter over time in aCSF, suggesting that degradation occurred primarily through surface erosion (Figure 5A,B, top panels). Scaffolds formed from fibers with quercetin displayed similar initial morphologies to those formed from fibers without quercetin, but over time, the fibers appeared to fuse together (Figure 5A,B, bottom panels). This behavior coincided with the appearance of larger pores in the material containing fibers loaded with quercetin. By day 7, in aCSF, a lattice-like network of PVA fibers was present for the scaffolds prepared with fibers loaded with quercetin.

Fiber swelling during the first 24 h of incubation reached values of up to 168.0 ± 3.5 and 152.6 ± 2.9% for fibers prepared with and without quercetin loading, respectively (Figure 5C). Maximum swelling of the material occurred after 3 days of incubation in aCSF for scaffolds formed from fibers, regardless of whether the scaffolds were formed from fibers containing quercetin. However, significant differences in swelling were observed from 8 h onward between fibers without quercetin and fibers containing quercetin. After 7 days in aCSF, the fibers without quercetin swelled 184.5 ± 3.3%, whereas the fibers containing quercetin swelled 199.4 ± 3.3%. These data suggest that incorporation of quercetin makes the scaffold more hydrophilic, leading to increased uptake of water and, potentially, increased degradation in aqueous media.

Quercetin Release from PVA Fiber Scaffolds

Scaffolds formed from PVA fibers loaded with quercetin were placed in aCSF to mimic the physiological fluid of the spinal cord and better understand the release profile of quercetin in nervous system tissue as a model for in situ application. UV/vis spectroscopy was used to quantify quercetin release into aCSF from the degrading scaffold over the course of 10 days (Figure 6). As expected, based on the swelling behavior of the material and its degradation profile, a burst release of quercetin corresponding to a cumulative release of ∼5% occurred in the first few hours of incubation in aCSF. Following this initial burst release, sustained release of quercetin was observed for several days. The release rate slowed between days 3 and 10, reaching a cumulative release of 56 ± 6% by day 10 in aCSF. The release profile followed a second order polynomial (R² > 0.99), demonstrating that the rate of quercetin release was greatest initially (t = 0), and decreased over time. This indicates that release did not follow zero order kinetics, where quercetin would be released at a constant rate until depleted. It is possible that quercetin molecules furthest from the surface of the fiber may simply take longer to diffuse into the aCSF than quercetin molecules closer to the surface of the fiber. It is also possible that release is not solely directed by quercetin, indicating some degree of interaction between quercetin and the PVA polymer chains affecting quercetin release.

Figure 6.

Cumulative release of quercetin in aCSF. Glyoxal-crosslinked PVA scaffolds with the addition of 200 μM of quercetin to the one-pot reaction were assessed. Error bars represent 90% confidence intervals with respect to the mean (n = 3).

As quercetin is known to mediate its neuroprotective effects by limiting oxidative damage, burst release would be beneficial to prevent excessive foreign body reactions and inflammation after implantation.^61,62 However, a persistent inflammatory response may lead to fibrosis and scarring, which may result in either misguided growth of regenerating axons or a lack of regeneration.⁶³ Therefore, sustained release of quercetin may also help mitigate these effects over a longer period. Although there is only one comparable study to have examined quercetin release from a PVA fiber scaffold,²⁹ there are precedents for the loading and release of other small molecule drugs from fibrous scaffolds with antioxidant and anti-inflammatory properties. For example, ibuprofen has been loaded in polytrimethylene carbonate-co-ε-caprolactone electrospun fibers to limit inflammation and promote axonal regeneration following nerve injury.⁶⁴ In another example, melatonin was loaded into electrospun polycaprolactone nerve scaffolds to limit oxidative stress and inflammation.⁶⁵ Ibuprofen was almost completely released from the polytrimethylene carbonate-co-ε-caprolactone fibers after 8 h in PBS, whereas the melatonin was released from the polycaprolactone scaffolds in a sustained manner.^64,65 Although no explicit release studies were completed, antioxidant activity assays and reduced reactive oxygen species at 6 and 12 weeks in rat models demonstrated improved functional recovery following nerve injury.⁶⁵

In addition, based on previous reports of quercetin contributing to differentiation and neurite formation in PC12 cells,^58,59 we hypothesized that sustained quercetin release would lead to improved PC12 cell differentiation when combined with fibers that support the anisotropic growth of adherent cells.^37,39 Thus, PC12 cells were cultured on substrates containing PEO-Col fibers only, PEO-Col/PVA fibers, and PEO-Col/quercetin-loaded PVA fibers. 30 min after cell seeding on PEO-Col fibers, PC12 cells exhibited rounded morphologies but appeared to interact with the PEO-Col fibers as loose aggregates (Figure 7A). Stable attachment to the PEO-Col fibers appeared to be NGF-dependent (Figure 7B,C), with NGF-treatment at day 3 leading to the differentiation of the PC12 cells into cells with neuronal morphologies for the PEO-Col, PEO-Col/PVA, and PEO-Col/quercetin-loaded PVA fibers by day 10 in culture. The MTT assay revealed that, regardless of treatment condition, cells displayed similar levels of viability and metabolic activity at the 10 day time point (Figure S6). Qualitatively, PEO-Col/quercetin-loaded PVA fibers promoted the most robust differentiation response of any of the conditions, as indicated by the presence of numerous elongated cells with neurite-like processes (Figure 7C), thus supporting the potential to use fibrous scaffolds formed from these materials to promote nerve regeneration.

Figure 7.

Substrates prepared from PEO-Col and quercetin-loaded PVA fiber support the differentiation of PC12 cells. (A) PC 12 cells start to associate with the PEO-Col fibers within 30 min of cell seeding. (B) NGF is required for the differentiation of PC12 cells on the PEO-Col fibers. Cells that were not exposed to NGF remained rounded over the course of 10 days of culture. (C) PC12 cells cultured on PEO-Col fibers in the presence of NGF and quercetin-loaded PVA fibers displayed the most robust neurite extension of the conditions examined. Arrows indicate the direction of fiber alignment. PEO Col denotes the presence of collagen fibers, and PVA Qu denotes the presence of quercetin-loaded PVA fibers. Scale bars are 100 μm.

Summary and Outlook

Since PVA is a hydrophilic polymer that is readily soluble in aqueous environments, it requires crosslinking in order to be useful for drug release.^31,35,50 Here, glyoxal in a one-pot reaction was demonstrated to promote crosslinking of contact-drawn PVA fibers. Glutaraldehyde is one of the most widely used crosslinking agents, allowing PVA to be processed into microspheres, hydrogels, and electrospun scaffolds for drug release.^31,66,67 Despite its popularity, glutaraldehyde may not be an optimal crosslinker for PVA since it produces a crosslink of greater length in comparison to the glyoxal crosslink. This may lead to steric hindrance during a one-pot reaction and may also lead to increased susceptibility to degradation. Glyoxal is the simplest dialdehyde molecule and an inexpensive crosslinker that forms acetal linkages between the free hydroxyl groups of PVA under mild reaction conditions.⁵⁰ Crosslinking of PVA with small aldehydes is likely to result in a densely packed network of PVA molecules in the fiber matrix, thereby aiding in the retention and sustained release of small molecules loaded into the material.⁶⁸ Glyoxal can also be easily quenched with free amines or alcohols, providing a convenient mechanism to control the crosslinking reaction and prevent unintended crosslinking of native proteins present in biological materials.^69,70

Although electrospinning is the most widely used approach for fabricating fibrous scaffolds, there are other emerging techniques for preparing drug loaded fibers for nerve repair. For example, polylactic-co-glycolic acid scaffolds for supporting Schwann cell growth have been fabricated through melt spinning,⁷¹ and polycaprolactone nanofibers have been fabricated through centrifugal spinning⁷² for use in an anisotropic alginate hydrogel to enhance the differentiation of entrapped stem cells into neuronal cells. In general, electrospinning of PVA can produce finer fibers (∼50 nm to 2 μm) than contact drawing (∼2–4 μm), with larger electrospun fiber diameters formed for higher concentrations and molecular weights of PVA.^73,74 Centrifugal and wet spinning tend to form even larger fibers, with fiber diameters ranging from ∼0.6 to 3 μm for centrifugal spinning⁷⁵ and up to ∼40–60 μm for wet spinning.⁷⁶ Each fiber forming technique has distinct ways to modify the fiber diameter based on modifications to spinning apparatuses and fabrication protocols, so these ranges are only provided as reference points. That said, the use of nozzles in these other systems generally leads to complicated setups and a resulting decrease in the throughput of fiber fabrication and processing. The contact drawing process does not use nozzles or complex collection systems and, therefore, may be more amenable to manufacturing at scale.

Previous work focused on electrospun PVA–gelatin–gellan mats loaded with quercetin serves as a basis for comparison of the electrospinning and contact drawing approaches and demonstrates the utility of quercetin-loaded fibers for tissue engineering applications.²⁹ In terms of similarities, both processes allow efficient incorporation of quercetin during the fiber formation step, and both employ PVA as the main polymeric component of the fiber. However, Vashisth et al. included both gelatin and gellan in their fibers to improve stability and did not include a crosslinking agent such as glyoxal during the fiber manufacturing process,²⁹ presumably because of issues associated with clogging of the electrospinning nozzle. Furthermore, the PVA–gelatin–gellan system utilized a supporting hydrogel to create the final scaffold material. Despite these differences in material composition, the PVA–glyoxal system displayed a comparable degradation rate to the PVA–gelatin–gellan system (∼30% over 7 days). Thus, it may be possible to further tune the degradation profile of the glyoxal-crosslinked fibers be incorporating them into a hydrogel, following a similar approach to that described by Vashisth et al.²⁹ Although the degradation profiles of the two materials were similar, there were important differences with respect to quercetin release. The PVA–glyoxal system displayed a modest burst release, followed by sustained release over 10 days to a total of 50% cumulative release, compared to approximately 60% release by burst in the first 24 h for the PVA–gelatin–gellan system, followed by 30% sustained release over days 1–7 (90% total over 7 days). The longer duration of sustained release of quercetin from the PVA–glyoxal system might be a result of the crosslinking reaction, potentially tethering the quercetin to the scaffold. Thus, by exploring a combination of the two approaches, it may be possible to further tune the quercetin release profile as desired for nerve repair or other clinical indications. Finally, it should be noted that the work by Vashisth et al. suggests that PVA fibers do not adversely influence the growth of neural cells, as demonstrated with the SH-SY5Y cell line, an observation supported here by experiments culturing PC12 cells on substrates with PEO-Col and PVA fibers.

In conclusion, the PVA fiber scaffolds presented here provide a versatile platform for loading small molecules that hold the potential to be used as a component of a multifunctional nerve guide. Through contact drawing, it is not only possible to produce large numbers of high-quality fibers in a short period of time under aqueous processing conditions but also to load the fibers with small molecule drugs during the fabrication process, eliminating the need for postprocessing steps required to incorporate drugs into the materials. From these contact-drawn glyoxal-crosslinked fibers, it is possible to achieve cumulative release of a small molecule drug (quercetin) over at least 10 days in aCSF, opening the door to many additional applications in drug delivery and tissue engineering. Future work will investigate the release kinetics of quercetin in vivo and explore additional fiber and additive formulations for modulating inflammation in soft tissue.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.2c00191.

• Schematic of the contact drawing apparatus for the analysis of single fiber formation; image processing steps to quantify quercetin fluorescence in hydrated PVA fibers; standard curve for quercetin content in artificial cerebrospinal fluid; fiber diameter as a function of pull speed, glyoxal concentration, and the presence of 200 μM quercetin in the one-pot reaction; and MTT assay for PC12 cells cultured on various fiber substrates (PDF)

Author Contributions

Z.B.V. and S.K.V. contributed equally to the work. Z.B.V. planned and conducted material fabrication and characterization experiments, analyzed the data, and wrote the first draft of the manuscript. S.K.V. performed the cell culture studies, analyzed the data, and contributed to drafting the manuscript and finalizing the text. J.K.R. and J.P.F. directed research activities, assisted with data analysis and interpretation of experimental data, and assisted with writing and editing the manuscript.

The authors declare the following competing financial interest(s): J.K.R. and J.P.F. own equity in 3DBioFibR Inc., a company commercializing advanced fiber technologies.