Controlled Release of Lysozyme Using Polyvinyl Alcohol-Based Polymeric Nanofibers Generated by Electrospinning

Abstract

Polymeric nanofibers generated via electrospinning offer a promising platform for drug delivery systems. This study examines the application of electrospun polyvinyl alcohol (PVA) nanofibers for controlled lysozyme (LZM) delivery. By using various PVA grades, such as the degree of polymerization/hydrolysis, this study investigates their influence on nanofiber morphology and drug-release characteristics. LZM-loaded PVA monolithic nanofibers having 50% drug content exhibit efficient entrapment, wherein rapid dissolution is achieved within 30 min. The initial burst of LZM from the nanofiber was reduced as the LZM content was lowered. The initial dissolution is greatly influenced by the choice of PVA grade used; fully hydrolyzed PVA nanofibers demonstrate controlled release due to the reduced water solubility of PVA. Furthermore, coaxial electrospinning, which creates core–shell nanofibers with polycaprolactone as a controlled release layer, enables sustained LZM release over an extended period. This study confirms a correlation between PVA characteristics and controlled drug release and provides valuable insights into tailoring nanofiber properties for pharmaceutical applications.

Introduction

Research on the application of polymeric nanofibers generated by electrospinning as a drug delivery system (DDS) has attracted much attention recently.¹⁾ The electrospinning method is a technology that forms nanofibers with fiber diameters ranging from tens to hundreds of nanometers by applying a high voltage (over 10 kV) to a polymer solution. In conventional single-nozzle electrospinning, when a high voltage is applied, the charge on the metal nozzle is transferred to the liquid, which accumulates on the droplet surface, and deforms the droplet at the tip from a sphere to a cone (this conical droplet is known as a Taylor cone). When the electrical attraction overcomes the surface tension of the droplet, the solution is stretched from the tip of the Taylor cone to the collector side (grounded side or opposite charge side) and sprayed in a straight line for approximately 25 mm. Thereafter, the fiber elongates in a spiral trajectory (swirling like a whip), and simultaneously, its diameter decreases to a nanosized (submicron size) to reach the collector. The solvent evaporates during the swirling process, and a dry sheet-like film (nanofiber sheet) is collected by the collector. Electrospinning as a process is characterized by its capability to continuously generate nanofibers from liquid raw materials under low energy and ambient temperature conditions. Thus, electrospinning is a promising formulation method for thermally unstable new modalities, such as biopharmaceuticals and nucleic acid medicines.²⁾

By using lysozyme (LZM) as a model biopharmaceutical, we attempted to control drug release by polymeric nanofibers in this study. Water-soluble polyvinyl alcohol (PVA), approved as a general pharmaceutical excipient, was used as the substrate for the LZM-containing nanofibers. The degree of hydrolysis of PVA (the ratio of hydroxyl/acetyl groups) and the degree of polymerization (the number of monomers forming a polymer) govern its physical properties, notably its water solubility.^3–5)

In general, PVA is dissolves well in water because of the large number of hydroxyl groups in the molecule that interact with water molecules via hydrogen bonds. The solubility of PVA is likely to decrease as the degree of hydrolysis increases. Fully hydrolyzed PVA has a lower solubility in water because the intermolecular and intramolecular hydrogen bonds formed between the hydroxyl groups of the PVA molecules are larger than the interactions with water. Therefore, fully hydrolyzed PVA with high crystallinity requires more energy to dissolve than partially hydrolyzed PVA. The partially hydrolyzed PVA molecule has more hydrophobic acetate groups, which reduces hydrogen bonds between PVA molecules owing to steric hindrance. Consequently, interactions between partially hydrolyzed PVA molecules and water molecules increase, resulting in increased solubility. Regarding the degree of polymerization of PVA, PVA with a lower molecular weight dissolves in water faster than PVA with a higher molecular weight owing to less intramolecular interaction.⁶⁾ Table 1 lists the PVA grades used in this study.

Table 1. Polyvinyl Alcohol (PVA) Grades with Different Degrees of Polymerization and Hydrolysis Used to Prepare LZM-Containing Nanofibers

Abbreviation	Trade name (GOHSENOL™ series)	Degree of polymerization	Degree of hydrolysis (mol%)
PVA24-88	EG-40P	2400	88
PVA6-88	EG-05P	600	88
PVA25-80	KH-20	2500	80
PVA17-98	NH-18	1700	98

In the case where the substrate of the nanofibers is a water-soluble polymer such as PVA, water can be used as the solvent of the starting solution for the electrospinning process.⁷⁾ Drugs that are water-soluble, such as peptides and proteins, can be simply dissolved in an aqueous polymer solution for loading drugs onto the nanofibers. In this study, we attempted to apply high lysozyme loading to PVA nanofibers.

Achieving sustained drug release using nanofibers composed of water-soluble polymer PVA is expected to be more challenging than that from nanofibers made from biodegradable hydrophobic polymers such as poly(lactic-co-glycolic acid) (PLGA) and poly(ε-caprolactone) (PCL).^8,9) Different grades of PVA having varying degrees of hydrolysis and polymerization could control the solubility of PVA nanofibers; however, there are few reports on this. By using the PVA grades, LZM release from various PVA nanofibers was evaluated in this study, as shown in Table 1.

Finally, electrospinning with a coaxial nozzle was conducted to achieve sustained LZM release from the PVA nanofibers over a longer period of time. Core–shell nanofibers coated with PCL as a controlled release layer in PVA/LZM nanofibers were prepared to generate long-term sustained release nanofiber formulations based on diffusion of the shell layer.

Results and Discussion

Effect of the PVA Grades on LZM Release from Nanofibers

LZM-containing PVA monolithic nanofibers were first prepared by electrospinning using various grades of PVA (Table 2). The weight ratio of LZM to PVA was one: one in order to obtain nanofibers with high drug content. In F1–F3 nanofibers, scanning electron microscope (SEM) images revealed that PVA nanofibers with 50% LZM content can be prepared, as shown in Figs. 1a–c. Figure 1d shows the fiber diameter calculated from each SEM image. The fiber diameters of the nanofibers in F1 and F3 were approximately 300–350 nm. In case of PVA6-88 (F2), which had a small degree of polymerization, the fiber diameter was the thinnest, and some beads were observed because the viscosity of the PVA6-88 solution tended to be low.

Table 2. Composition of LZM-Containing Nanofibers Prepared by Partially Hydrolyzing PVA by Electrospinning

Formulation	PVA grade	PVA/LZM (w/w)	PVA (g)	LZM (g)	Distilled water (g)
F1	PVA24-88	1/1	4	4	46
F2	PVA6-88	1/1	10	10	40
F3	PVA25-80	1/1	4	4	46
F4	PVA24-88	2/1	4	2	46
F5	PVA24-88	3/1	4	1.3	46
F6	PVA25-80	2/1	4	2	46
F7	PVA25-80	10/1	4	0.4	46
F8	PVA25-80	20/1	4	0.2	46

Fig. 1. Characterization of LZM-Containing PVA Monolithic Nanofibers with Different Grades of PVA (Weight Ratio: LZM/PVA = 1/1)

Scanning electron microscope (SEM) images of LZM-containing PVA nanofibers F1 (a), F2 (b), and F3 (c) (scale bar, 5 µm). (d) Average fiber diameter measured by Image J from each SEM image. (e) LZM dissolution profiles of LZM-containing PVA nanofibers. Values are expressed as the mean ± standard deviation (S.D.) of the mean (n = 3).

We completely dissolved F1 nanofibers in water and then measured the entrapment efficiency of LZM at an absorbance of 281 nm, which was found to be 101.4 ± 0.2%. This high drug encapsulation efficiency in electrospun fibers is likely due to both the drug and polymer being soluble in the solvent (water), and minimal drug loss during the encapsulation process.¹⁰⁾ The absence of LZM inactivation in PVA nanofibers following the electrospinning process was confirmed via the LZM Activity Kit¹¹⁾ (Supplementary Fig. 1).

To the best of our knowledge, the drug loading capacity into drug carriers such as liposomes and polymer nanoparticles containing drugs has been around 10 wt% at the highest.¹²⁾ On the other hand, a remarkable feature of electrospinning was that a 50 wt% payload content in a submicron-sized space could be easily achieved in a single step.¹³⁾ During manufacturing, transport, and storage under environmental stresses, biopharmaceuticals with complex structures, especially their liquid dosage forms, may degrade. Therefore, solid formulations of biopharmaceuticals are frequently required to ensure stability. Lyophilization and spray-drying are used universally to dehydrate biopharmaceuticals; however, in some cases, they do not provide sufficient gentle drying and may deactivate some biopharmaceuticals.¹⁴⁾ Electrospinning rapidly evaporates solvents at room temperature and atmospheric pressure, making it a candidate for gentle drying techniques that can be used as an alternative to freeze-drying or spray-drying.¹⁵⁾

The SEM images of F1 nanofibers, which contain a 1/1 (w/w) of PVA/LZM, stored for four weeks under accelerated conditions are presented in Supplementary Fig. 2. When compared to the initial images, a slight increase in fiber diameter and some changes in fiber shape were observed after four weeks. It is important to note that, while PVA nanofibers typically exhibit good stability,^7,16) the observed alterations in this study might be attributed to the high content of LZM within the nanofibers, which could affect their stability. Although water content was not measured, the nanofibers might have absorbed moisture under humid conditions, further influencing their physical properties. In this research, evaluations such as dissolution tests were conducted within 1–2 d after the preparation of the nanofibers. Given these observations, future storage of LZM-containing PVA nanofibers may require moisture-proof packaging, such as aluminum wrapping or the inclusion of silica gel within the packaging container, to mitigate potential stability issues.

The dissolution of LZM from these nanofibers (F1–3) was tested (Fig. 1e). For all nanofibers, approximately 100% dissolved within 30 min after starting the dissolution test. The LZM dissolution of F2 was the fastest, which may be attributed to the water solubility of PVA6-88 itself rather than to its small fiber diameter. Conversely, F3, which is composed of low hydrolysis PVA, suppressed initial dissolution the most, albeit by a small difference. The wettability of the nanofibers in water was considered to be slightly reduced by the use of PVA25-80, which contained more vinyl acetate groups.

Effect of the PVA/LZM Ratio on LZM Release from Nanofibers

The effect of LZM content on drug-release behavior was evaluated for nanofibers based on PVA24-88 (Fig. 2a) and PVA25-80 (Fig. 2b), which have different degrees of hydrolysis. In both PVAs, the rate of LZM dissolution from the nanofibers tended to decrease as the LZM content decreased. It was suggested that the excess PVA in the nanofibers took longer to dissolve in water, which thus reduces the initial dissolution of LZM. The LZM content was reduced to a PVA/LZM ratio of 10/1 or 20/1 for the relatively slow dissolution PVA25-80 nanofibers, with no significant change in the LZM release properties. In the case of PVA nanofibers, the release mechanism of LZM depends on the dissolution rate of PVA itself, suggesting that the solubility of PVA in nanofibers does not change when the LZM content is below a certain level.

Fig. 2. Effect of the PVA/LZM Ratio on LZM Dissolution from PVA Monolithic Nanofibers with Different Degrees of Hydrolysis

a) PVA24-88 (EG-40P) and b) PVA-25-80 (KH-20) nanofibers. Values are expressed as the mean ± S.D. of the mean (n = 3).

LZM Nanofibers Containing Fully Hydrolyzed PVA

As shown in Fig. 2, there was a possibility of controlling LZM release from nanofibers via LZM content; however, partially hydrolyzed PVA had high solubility in water, which limited controlled release for a long time. Therefore, we attempted in this section to prepare PVA nanofibers with high LZM content using PVA17-98, a fully hydrolyzed PVA with relatively low solubility.

Table 3 shows the formulation of nanofibers incorporating completely hydrolyzed PVA. An aqueous solution of PVA17-98 and LZM dissolved in 1/1 (w/w) was prepared; however, the nozzle tended to clog during the electrospinning process and nanofibers could not be generated in a stable manner because of its very high viscosity. It is possible that completely saponified PVA could be spun by optimizing the PVA/LZM solution concentration and solvent; however, this was not done in this study.

Table 3. Composition of LZM-Containing Nanofibers Incorporated with Fully Hydrolyzed PVA by Electrospinning

Formulation	PVA24-88/PVA17-98 (w/w)	PVA24-88 (g)	PVA17-98 (g)	LZM (g)	Distilled water (g)
F9	3/1	1.8	0.6	0	20.2
F10	3/1	1.8	0.6	2.4	22.6
F11	2/1	1.5	0.6	0	20.5
F12	2/1	1.5	0.75	2.25	22.75

We attempted to prepare LZM nanofibers by blending fully hydrolyzed PVA with partially hydrolyzed PVA to incorporate PVA17-98 into the nanofibers. Nanofibers with PVA24-88/PVA17-19 weight ratios of 3/1 and 2/1 were prepared. In the absence of LZM (F9 and F11), SEM images confirmed that nanofibers with uniform diameters could be prepared at both ratios (Figs. 3a, b). Nanofiber mats could be recovered when LZM was incorporated (F10 and F12); however, the fibers became thicker than those without LZM, and some of them were observed to fuse with the fibers (Figs. 3c, d). This is probably because of the higher viscosity of the raw material solution caused by the addition of LZM. Cumulative drug release at 30 min following dissolution testing with F10 and F12 LZM nanofibers is shown in Fig. 3e. F10 and F12 with completely hydrolyzed PVA, which is less soluble in water, achieved controlled initial dissolution compared with F1.

Fig. 3. LZM-Containing PVA Monolithic Nanofibers Using a Blend of Fully Hydrolyzed (PVA17-98) and Partially Hydrolyzed PVA (PVA24-88) with a PVA/LZM Weight Ratio of 1/1

SEM images of a) F9 and b) F11 nanofibers without LZM and c) F10 and d) F12 nanofibers containing LZM (scale bar, 5 µm). e) LZM dissolution percentage from nanofibers 30 min after the start of the dissolution test. Values are expressed as the mean ± S.D. of the mean (n = 3).

PCL/PVA Core–Shell Nanofiber to Release LZM over the Long Term

In the previous section, we demonstrated that the release of LZM from PVA nanofibers could be controlled by different grades of PVA and LZM content. However, controlling long-term release with PVA alone would be challenging because LZM release from nanofibers depends on the solubility of PVA itself. We attempted to coat PVA/LZM nanofibers with hydrophobic PCL using coaxial needle electrospinning to achieve extended LZM release.

The preparation of core–shell nanofibers generally requires that the polymer solution for the core layer be of lower viscosity than that for the shell layer to stretch the core layer.⁹⁾ Hence, the PVA24-88 concentration in the PVA/LZM solution pumped to the inner needle was 2%, which had lesser viscosity than the 8% concentration used in F2. SEM and negatively stained TEM images of PCL/PVA core–shell nanofibers are shown in Figs. 4a and b. The SEM images revealed that the fabricated fibers were approximately 1 µm in diameter, and the TEM image confirmed the core–shell structure inside the nanofibers. The contrast in the TEM image confirmed the boundary between the PVA/LZM and PCL layers.

Fig. 4. Characterization of LZM-Containing PCL/PVA Core–Shell Nanofibers Prepared by Coaxial Electrospinning

a) SEM image (scale bar, 5 µm) and b) TEM image of PCL/PVA core–shell nanofibers (scale bar, 0.5 µm). The white dotted line in the TEM image is the boundary of the core–shell layer. c) Dissolution test of LZM from nanofibers, comparing the dissolution profiles of F10 nanofibers and PCL/PVA core–shell nanofibers. Values are expressed as the mean ± S.D. of the mean (n = 3).

As shown in Fig. 4c, the LZM dissolution test from core–shell nanofibers was performed. F10 nanofibers containing completely hydrolyzed PVA exhibited relatively slow dissolution compared with other PVA-alone formulations; however, nearly 80% LZM dissolution was observed after 1 h. Furthermore, F10 incorporated partially hydrolyzed PVA, which led to the dissolution of PVA24-88 in the fiber and the release of LZM as an initial burst. The subsequent gradual dissolution of PVA17-98 in the nanofibers may have released the LZM remaining in the nanofibers.

Conversely, core–shell nanofibers demonstrated sustained release of LZM for >3 d. Nanofibers with a shell layer of hydrophobic PCL remained in the vessel following the dissolution test. The LZM dissolution profile followed the Higuchi model (Supplementary Fig. 3), suggesting that release is controlled via drug diffusion in the PCL matrix of the shell layer.^17,18) During the 3-d period, the mechanism for erosion of the PCL shell layer may have a limited effect on the release of LZM.

While LZM was used as a model drug in this study, the nanofiber-based technology capable of controlled release, depending on the PVA grade as well as the combination of PVA and PCL, can be applied beyond LZM to encapsulate biologics, proteins, enzymes, peptides, and nucleic acids into PVA nanofibers through electrospinning.^2,19) These nanofibers, specifically designed for encapsulating biologics and capable of regulating their release, offer significant potential in a range of applications.²⁰⁾ Primarily, they are well-suited for tissue engineering and regenerative medicine as scaffold materials, as well as for transdermal therapeutic systems and wound dressings. Furthermore, their tailored use for site-specific drug release in the gastrointestinal tract via oral administration,²¹⁾ in mucosal vaccines such as sublingual applications,²²⁾ and for localized biopharmaceutical treatments in mucosal tissues like the eyes,²³⁾ lungs,²⁴⁾ and vaginal areas,²⁵⁾ demonstrates their versatility and effectiveness.

The association between PVA grade and controlled release of therapeutic payloads from nanofibers confirmed in this study could be a significant finding for the development of pharmaceutical nanofibers for various anticipated applications.

Conclusion

One-step preparation of PVA nanofibers with high content of LZM in the 200–400 nm range by electrospinning was achieved easily. The degree of polymerization of PVA impacted morphology of the nanofiber, whereas the degree of hydrolysis of PVA and LZM content affected the release of LZM from the nanofibers. Incorporation of completely hydrolyzed PVA into the nanofibers led to reduced water solubility of the nanofibers; thus, PVA solubility-dependent control of LZM release from the nanofibers was achieved. The preparation of PVA nanofibers coated with PCL (core–shell nanofibers) via coaxial electrospinning permitted the long-term control of LZM release via the LZM diffusion mechanism in the PCL layer matrix. Nanofibers that contain biopharmaceuticals such as LZM can be generated by electrospinning with PVA as a base material for nanofibers, and drug release could be achieved by modifying the pharmaceutical formulation to achieve the desired drug release.

Experimental

Materials

Mitsubishi Chemical Corporation (Tokyo, Japan) provided the PVAs (GOHSENOL™ series). Table 1 presents the degree of polymerization and hydrolysis of the PVAs used in this study. PVAs are abbreviated according to the degrees of polymerization and hydrolysis; for example, PVA24-88 has a 24 × 10² degree of polymerization and 88% degree of hydrolysis. LZM from Egg White was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). N,N-Dimethylformamide (DMF) and chloroform were purchased from Kishida Chemical Co., Ltd. (Osaka, Japan). Polycaprolactone (Mn = 80000) was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). All other chemicals were of the highest grade available commercially.

Preparation of LZM-Loaded Polymeric Nanofibers via Electrospinning

For the electrospinning process, the LZM-containing PVA was loaded into a syringe via a 22G nonbeveled needle (Terumo, Tokyo, Japan). Randomly oriented nanofibers were electrospun by applying 10 kV to the needle with a high-voltage supplier (HVU-30P100, MECC CO., LTD., Fukuoka, Japan). The standing plate collector was located 12 cm from the needle, and by using a syringe pump, the solution was loaded at a feed rate of 0.5 mL/h (Yutaka Electronics Manufacturing, Gifu, Japan). For the preparation of LZM-containing PVA/PCL core–shell nanofibers, the shell solution was prepared by dissolving 1.2 g of PCL in 10 g of chloroform/DMF (4/1) solution. The core solution was prepared by dissolving 0.2 g of PVA EG-40P in 9.8 g of distilled water, followed by complete dissolution of 0.1 g of LZM. The core and shell solutions were filled into separate 5 mL syringes; a coaxial spinneret with a 26-gauge core needle and a 20-gauge shell needle was connected to the syringe with a PFA tube. By using two syringe pumps, the core solution was pumped at a flow rate of 0.2 mL/h and the shell solution at a flow rate of 0.5 mL/h. A potential of 18 kV was applied to the coaxial spinneret. The distance between the spinneret and plate collector was 12 cm.

Physicochemical Properties of the LZM-Loaded Polymeric Nanofibers

The nanofiber mat prepared by electrospinning was imaged using a scanning electron microscope (SEM, JSM-6510LV, JEOL, Japan). The fiber diameter (mean and standard deviation) was calculated by measuring 100 randomly selected locations from SEM images using image analysis software (ImageJ™, NIH). PVA/PCL core–shell nanofibers were observed by transmission electron microscopy (TEM, H-7000; Hitachi, Tokyo, Japan) on negatively stained specimens.

For determining the LZM content in PVA nanofibers, nanofibers were completely dissolved in water and filtered through a 0.45 µm cellulose acetate membrane. LZM content was determined by measuring absorbance at 281 nm using a UV-1800 UV–VIS spectrophotometer (Shimadzu, Kyoto, Japan). By using a Micrococcus lysodeikticus-based turbidimetric assay (Lysozyme Activity Kit, Sigma-Aldrich), LZM activity was determined.

Dissolution Test

Dissolution tests of LZM from polymeric nanofibers were performed using the paddle method described in the Japanese Pharmacopoeia (JP 17th Edition). A sample containing 15 mg LZM was added to 900 mL distilled water at 37 ± 0.5 °C under paddle stirring at a rotation speed of 50 rpm. The nanofibers were loaded into the dissolution test vessel using an alternative sinker as specified in JP17. At specific time intervals, 5 mL samples were withdrawn and filtered through a 0.45 µm cellulose acetate filter. By measuring absorbance at 281 nm using a UV-1800 UV-VIS spectrophotometer, the LZM concentration in the collected samples was determined.

Acknowledgments

The Laboratory of Nanofiber Technology (Gifu Pharmaceutical University) is an endowment laboratory supported by an unrestricted Grant from Mitsubishi Chemical Corporation.

Conflict of Interest

KT reports financial support provided by Mitsubishi Chemical Corporation. KH, AK, NY, and YT report a relationship with Mitsubishi Chemical Corporation that includes employment. KH, AK, NY, YT, and KT have patent #Japanese Patent Application No. 2022-012541 pending to Mitsubishi Chemical Corporation.

Supplementary Materials

This article contains supplementary materials.

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