2015 Volume 63 Issue 2 Pages 81-87
In this study, we applied an electrospinning (ES) method, which is mainly employed in the textile industry, to the field of pharmaceuticals. We developed and modified an ES instrument and then utilized it to produce methacrylic acid copolymer S (MAC) nano-fibers to prepare tablets. By attaching a conductor rod made from stainless steel to the central part of the nano-fiber-collection plate of the ES apparatus, a MAC nano-fiber sheet could be produced effectively. In addition, we studied various operating conditions for this new ES method, including needle gauge, voltage between the electrodes, distance between the needle and nano-fiber-collection plate and the flow rate of MAC polymer solution, but these had no significant effect on the diameter of MAC nano-fibers. On the other hand, the viscosity (concentration) of MAC polymer solution and permittivity of solvent used to dilute MAC were closely related to the mean diameter of the nano-fibers. Tableting of MAC nano-fibers was performed using a tableting machine without lubricants, and addition of Tween 20 to the tablets enabled regulation of the release profile of a water-soluble drug. The modified ES method reported here is a useful technique for the controlled-release of drugs and has wide-ranging potential for pharmaceutical applications.
Electrospinning (ES) works by applying a high voltage to a capillary tip and allowing a polymer solution to flow out. This technology can form nano-fibers by vaporizing the solvent to make the polymer solution form a superfine thread.1) Essentially, this technology has been actively developed and studied in the textile industry. In the fiber formation step from the polymer using the solvent-based ES method, the solvent is rendered volatile by electrical repulsion and fiber formation proceeds.2) In addition, the ES method is capable of production of nano-fibers at room temperature. The ES method, therefore, allows nano-fibers to be made from materials that are vulnerable to heat degradation, such as proteins.
The ES method has been widely studied as a process for making nano-meter sized non-woven fiber mats in the textile industry.3) A polymer solution, when electrostatically charged, can produce non-woven polymeric fibers of a desired size range by controlling the physical conditions of the polymer content, flow rate of the polymer solution or voltage between the electrodes, among other parameters.4,5) However, the amount of polymer solution extruded is extremely small; therefore, productivity is low. In addition, spherical beads can form on occasion, possibly due to instability in the ES state.6) Thus, it is clear that the ES method has a number of problems that remain to be solved. However, nano-fibers by the ES method have a lot of potential in the pharmaceutical field. It has been considered that nano-fibers by ES method can easily form a network structure and provide a sustained-release effect of several drugs. Presently, we are investigating drug release from a polymer matrix as a support medium in the formulation. Methacrylic acid copolymer S (MAC) is a representative example of a synthetic macromolecule that is widely used as an enteric-coating agent for making oral solid-type pharmaceutics. An anionic polymer is able to disintegrate in the intestine with pH-value of 7.0 to 8.0.7) In addition, we predicted that a nano-fiber of MAC could be an adequate alterative to support media used to regulate drug release from oral solid-type pharmaceutics. Despite the ES method having been used to make nano-fibers for a long time, the application of this technique for preparing medicinal products remains to be investigated.
In this study, utilizing a solvent-based ES method, which is mainly employed in the textile industry, we studied various ES conditions using MAC as a base polymer. In addition, we prepared nano-fiber-based tablets including uranine (UN) as a controlled-release drug delivery system, and evaluated in vitro dissolution.
MAC (EUDRAGIT® S100) was kindly supplied by Evonik Degussa Japan Co., Ltd. (Tokyo, Japan). UN (fluorescein sodium, Log Pow=0.10) was purchased from Nacalai Tesque (Kyoto, Japan). Polyoxyethylene sorbitan monolaurate (Tween 20, HLB=16.7) and sorbitan monolaurate (Span 20, HLB=8.6) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals and reagents were of analytical grade and were used without further purification.
Preparation of Test SolutionsMAC test solutions were prepared by diluting MAC with acetone, ethanol or methanol to a final concentration of 10–15% (w/v). As preparation for use, Tween 20 or Span 20 was added as a surfactant in the MAC test solution to a final concentration of 1–4% (w/v). In this study, UN, a highly water-soluble drug, was used as a model drug, and a standard stock solution of UN was prepared by dissolving in 66.7 mM phosphate buffer (pH=6.8) to a final concentration of 50 µg/mL, and then stored at 4°C in the dark. Working standards for a calibration curve of UN were prepared by diluting the standard stock solution to various concentrations. The polymer solution including UN was prepared by mixing MAC in methanol and the standard stock solution of UN at a volume ratio of 1 : 10. The final concentrations of UN and MAC in this mixture were 1.0% (w/v) and 10% (w/v), respectively.
Solvent-Based ES MethodNano-fibers were prepared by a solvent-based ES method. The ES experimental equipment consisted of a syringe pump, a stainless-steel needle (14G to 19G) and a high-voltage supplier, which was purchased from Kato Tech Co., Ltd. (Kyoto, Japan). Figure 1 shows a schematic illustration of the ES apparatus.
The ES method applies high voltage to a capillary tip and allows a polymer solution to extrude and form nano-fibers. a) Overall structure of the device; b) nano-fiber-collection plate; c) composition of a standard nano-fiber-collection plate; d) nano-fibers after extrusion of 10% (w/v) methacrylic acid copolymer S acetone solution at a voltage of 25 kV and a rate of 10 mL/h; e) cross-section view of d).
The polymer solution was drawn into a plastic syringe with a stainless-steel needle, and the syringe was fixed at a designated position in the ES apparatus. A nano-fiber-collection plate made from copper was negatively charged, and a grounding wire connected to the nano-fiber-collection plate. In order to collect nano-fibers more easily, a conductor-rod (4.2 mmϕ×35 mm length) made from stainless steel was attached vertically to the center of the fiber-collection plate. Then, the electrical voltage was increased from 15 to 25 kV between the needle and the nano-fiber-collection plate. The syringe needle and the nano-fiber-collection plate had positive and negative charges, respectively. The distance between the nano-fiber-collection plate and syringe needle was set from 5 to 15 cm, and the polymer solution was pumped at a flow rate of 5 to 20 mL/h. Consequently, a nano-fiber formed from the stream of polymer solution in the electric field with evaporation of the organic solvent, and a self-assembled nano-fibrotic sheet was obtained on the anode plate.
Viscosity of the Polymer SolutionPolymer solution viscosities of 10–15% (w/v) of MAC in organic solvent were measured by means of a SV-10 viscometer (A&D Co., Ltd., Tokyo, Japan). Forty milliliter of polymer solution was transferred into a cube with stirrer at ambient temperature (22–24°C), and the viscosity of the polymer solution was measured by a pendular-pulse method.
Microscopic ObservationThe shape and surface characteristics of nano-fibers formed by the ES method were observed by scanning electron microscopy (SEM) (S-3400N, Hitachi, Tokyo, Japan). The nano-fiber was sputter-coated with Au/Pd using a vacuum evaporator and examined using a SEM at 0.1 kV accelerating voltage. To compare the diameters of nano-fibers, mean diameters were calculated by measuring the virtual length based on 100 random portions on the respective image.
Preparation of Tablets from a Nano-fiber Sheet Including UNUN tablets derived from a nano-fiber sheet were prepared by tableting directly with 50 mg of MAC nano-fibrotic sheet including UN (4.5 mg) and surfactant (1–4% (w/v)) using a HANDTAB-100 tableting machine (Ichihashiseiki Co., Ltd., Kyoto, Japan). After the nano-fiber sheet was set in the form, it was compressed at a force of 10 MPa to form tablets (90 mmϕ×1.5 mm). The final amount of UN in a tablet was 4.5 mg. As a positive control, a nano-fiber sheet without surfactant was prepared.
In Vitro Dissolution TestAn in vitro dissolution test was carried out in accordance with the Japanese Pharmacopoeia XVI with some modifications. Tablets were added to 300 mL of 66.7 mM phosphate buffer (pH 6.8) for a dissolution test with magnetic stirring at a rotation speed of 100 rpm at 37°C. Samples of 1.0 mL each were collected at 0, 10, 20, 30, 60, 90, 120, 180, 240, 360, 420 and 480 min before and after the beginning of the dissolution test, with the same volume of medium being replaced for each sampling point. Immediately, the concentration of UN in the samples was measured by fluorophotometry using a DTX 800 multimode detector (Beckman Coulter, Inc., Tokyo, Japan), at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The calibration curve of UN was linear and passed through the origin with correlation coefficients of 0.999 or over.
The ES method has been studied as a process for making nano-fibers in the textile industry, and has been studied in the several fields from the 1930s.2) Recently, the ES method has been applied for stents in coronary intervention for myocardial infarction, and for the creation of cell scaffold for regenerative medicine.8–10) Our interest in the use of ES method is based on its versatility for making nano-materials, which can be available for preparing controlled-release systems, for water absorbable or inabsorbable drugs. Several methods or devices to form nano-fibers using the ES method have been introduced in the field of pharmaceutical sciences. For instance, the rotary drum as a collection plate11) or solvent-free melt electrospinning method12) is utilized to prepare nano-fibers for the sake of controlled-release formulations of inabsorbable drugs. In our previous report, we showed that an enteric coating material, MAC can be formed into nano-fibers using the ES method, and demonstrated the utilities of MAC nano-fibers for the controlled-release systems of water-soluble or water-insoluble drugs.13) In order to enhance the delivery of drugs with limited absorption due to poor solubility/dissolution, approaches are being developed to improve the dissolution rates and solubility of drug molecules. These approaches include identification of water-soluble salts of parent drugs, preparation of stable amorphous drug formulations, inclusion of solubility-enhancing agents in the dosage form, and particle size reduction. Technologies to reduce drug particle size to sub nano-meter range are being applied to product development more frequently. The ES method is also considered as one of the technologies that can produce nano-sized drugs incorporated in polymeric nano-fibers. In vitro and in vivo studies have demonstrated that the release rates of drugs from these nano-fiber formulations are enhanced compared to those from original drug substance. This technology has the potential to be used for enhancing the oral delivery of inabsorbable drugs. However, it is a fact that there are many operating conditions to be determined due to the nano-fiber collecting apparatus, polymer, solvent, transpire voltage between electrodes and physicochemical properties between materials embedded. In this study, we studied the preparation of nano-fibers using MAC, which is used as an enteric coating or as the continuous phase of a solid dispersion,7) and examined what conditions are required for preparation of formulations using MAC nano-fibers.
As shown in Fig. 1, our ES apparatus was composed of a syringe pump, a stainless-steel needle (14G to 19G), a high-voltage supplier and a metallic collection plate made from copper (Fig. 1a). A plain metallic plate is used for the collection of nano-fibers in the textile industry.14) However, for pharmaceutical applications of nano-fibers, a sheet-like or spongiform nano-fiber is needed. Therefore, a metallic collection plate made from copper with an attached conductor-rod made from stainless steel, which was located in the center of the plate, could enable to efficiently collect nano-fibers by concentrating on a conductor-rod with guided electric force (Figs. 1b, c). Applying a prescribed voltage, nano-fibers had begun to be collected within a copper-mesh, and then gradually got to pitch a tent round a fixed conductor-rod to become a layer structure (Figs. 1d, e). Consequently, a nano-fiber sheet surrounding the metallic collection plate was obtained, and the nano-fiber sheet was easily peeled off from the collection plate (Fig. 1d). Accordingly, the device we developed here can suppress the spreading of fibers and formation loss on the nano-fiber-collection plate. Furthermore, by attaching the metal conductor-rod at the center of the plate, it was found that nano-fibers can be collected more efficiently than on a plain metallic plate. The yield of nano-fibers which was prepared by our device with a conductor-rod increased by about 1.7 times as compared to that without a conductor-rod.
Figure 2 shows the effects of ES operating conditions such as needle gauge, voltage between the electrodes, distance between the needle and nano-fiber-collection plate and flow rate of MAC solution on the diameter of the nano-fibers.
Methacrylic acid copolymer S (MAC) solution was dissolved with acetone at a final concentration of 10% (w/v). The conditions except for examination were fixed as follows: needle gauge; 16G, voltage; 25 kV, distance; 10 cm, flow rate; 10 mL/h. Each symbol with bar represents mean±S.D. of 100 determinations.
Throughout this experiment, a 10% (w/v) MAC solution was used. Keeping the flow rate at 10 mL/h, distance and voltage between electrodes were set at 10 cm and 25 kV, respectively. When 14G, 16G and 19G needles were used as spray needle, the mean diameters of nano-fibers in these conditions were 743±233, 585±166 and 683±251 nm respectively. There were no significant changes in the mean diameter of MAC nano-fibers using these needles (Fig. 2a). Next, the effect of working voltage on the diameter of nano-fibers was studied. The flow rate, distance and needle gauge were set at 10 mL/h, 10 cm and 16G, respectively. At 15, 20 and 25 kV, the mean diameters of nano-fibers were 467±179, 443±140 and 526±182 nm, respectively. There were no significant differences in the mean diameters of MAC nano-fibers using these voltage conditions (Fig. 2b). Figure 2c shows the effect of distance between the needle and the nano-fiber collection plate on the diameter of nano-fibers. The needle gauge, voltage and flow rate were set at 16G, 25 kV and 10 mL/h, respectively. At 5, 10 and 15 cm distances between the needle and nano-fiber-collection plate, the mean diameter of nano-fibers was 302±98, 526±182 and 271±47 nm, respectively. Even though there was no significant difference among them, it was found that a distance of 10 cm gave a slightly large diameter of nano-fiber than the other distances (Fig. 2c). In addition, the effect of the flow rate on the diameter of nano-fibers was tested, where the needle gauge, voltage and distance were set at 16G, 25 kV and 10 cm, respectively. At a flow rate of 5, 10, 15 and 20 mL/h, the mean diameter was 409±203, 414±191, 335±88 and 382±164 nm, respectively. There were no significant changes in the mean diameter of MAC nano-fibers using these four flow rate conditions (Fig. 2d). From these results, as shown in Fig. 2, the needle gauge, voltage, distance and flow rate were set at 16G, 25 kV, 10 cm and 10 mL/h, respectively in the next experiments, because these conditions provided no significant changes in the diameter of MAC nano-fibers. Then, the effects of viscosity of the MAC solution and/or its concentration on the diameter of nano-fibers were investigated. As shown in Fig. 3, the diameter of nano-fibers had a positive correlation to the viscosity or concentration of MAC solution.
Where, needle gauge, voltage, distance and flow rate were set to 16G, 25 kV, 10 cm and 10 mL/h, respectively. Each symbol with bar represents mean±S.D. of 100 determinations.
As for MAC concentration, the mean diameters at 10, 12, 12.5, 13.75 and 15% (w/v), which viscosity for each concentration were 7, 9.5, 13.4, 20.2 and 31 mPa⋅s, were 526±182, 701±207, 904±434, 1327±575 and 1464±714 nm, respectively (Figs. 3a, b). Because the concentration of MAC has a direct correlation with the viscosity of its polymer solution, it was considered that adjustment of the polymer concentration between 10% (w/v) and 15% (w/v) could regulate the diameter of MAC nano-fibers. When the viscosity or the concentration was high, the saturation rate of MAC in the organic solvent is more accelerated during the evaporation of organic solvent under conditions of constant voltage and solvent flow rate. Hence, the fiber diameter becomes to be thick.
Figure 4 shows SEM images of the nano-fiber sheet with changes in solution viscosity. These visual results clearly demonstrated that the diameters of nano-fibers increased with increasing viscosity (concentration) of the MAC polymer solution. As an explanation of this phenomenon, it was considered that solid component amount of MAC per volume increased after the organic solvent evaporated when the viscosity or the concentration was high. In other reports on the ES method, lower viscosity of polymer solution, increasing applied voltage and reduced flow rate were effective for making thin fibers.15–17) In the case of MAC, however, it was concluded that only the viscosity of the polymer solution had a significant influence of the diameter of nano-fibers among the factors tested.
The viscosities of MAC polymer solution were 7.0 (a), 9.5 (b), 13.4 (c), 20.2 (d) and 31 (e) mPa·s.
Essentially, as the affinity of polymer against the solvent increases, polymer chains can spread out in the solution and increase their solubility.18) Because it is considered that the boiling point and permittivity of the solvent are also factors that affect the diameter of MAC nano-fibers, acetone, ethanol and methanol were tested as polymer solvents for MAC. Figure 5 shows the effects of these three solvents on the diameter of MAC nano-fibers.
The MAC concentration in acetone, ethanol and methanol was set at 10% (w/v). Each bar represents mean±S.D. of 100 determinations. * p<0.05.
The concentration of MAC, needle gauge, voltage, distance and flow rate were set at 10% (w/v), 16G, 25 kV, 10 cm and 10 mL/h, respectively. Using acetone, methanol and ethanol as solvents, the mean diameters of MAC nano-fibers were 526±182, 1339±290 and 1889±304 nm, respectively. In addition, as physicochemical factors relating to the ES method: boiling point, conductivity and permittivity were also examined. The boiling points for acetone, methanol, and ethanol are 56.5°C, 64.7°C and 78.4°C, respectively. Moreover, the conductivities of these solvents are 1.0×10−5, 1.6×10−6 and 1.1×10−6 S/m, respectively, and the permittivity values are 20.7, 32.6 and 24.3 F/m, respectively.19) In the ES method, the solvent evaporates instantaneously and a dried fiber accumulates on the metal collection plate. Wannatong et al. reported that solutions with lower boiling points gave fibers larger diameters than with high boiling points.20) The boiling point of the solvent to dissolve a polymer, therefore, one of a determination factor which can affect the diameter of the nano-fibers of MAC. However, as shown in Fig. 5, there was no clear correlation between the boiling point of the solvent and the diameters of the nano-fibers. In contrast, the permittivity was positively correlated with the diameter. These observations suggested that the permittivity of the solvent strongly influences the diameter of MAC nano-fibers.
One of the aims of our study was to create new pharmaceutical formulations using MAC nano-fibers. We investigated whether the addition of surfactants, which are representative surfactants for pharmaceuticals, to MAC acetone solution affected the MAC polymer solution or the diameter of nano-fibers at various concentrations of surfactants.
| Surfactants | Concentration (% (w/v)) | Viscosity (mPa·s) | Diameter±S.D. (nm) |
|---|---|---|---|
| No surfactant (MAC solution) | 10 | 13.3 | 526±182.08 |
| Tween 20 (HLB=16.7) | 1 | 12.5 | 329±72.48* |
| 3 | 11.2 | 388±87.18* | |
| 4 | 10.8 | 332±88.01* | |
| Span 20 (HLB=8.6) | 1 | 11.7 | 272±67.71* |
| 2 | 11.4 | 309±55.55* | |
| 4 | 11.2 | 439±115.28* |
Values are expressed as the mean±S.D. of 100 determinations. * p<0.05 compared with 10% (w/v) MAC acetone solution. The final concentrations of Tween 20 and Span 20 in polymer solutions were 1%, 3%, 4% and 1%, 2%, 4%, respectively.
As for Tween 20, which has a higher HLB value, the mean diameters of nano-fibers were 329±73, 388±87 and 332±88 nm, respectively, with 1%, 3% and 4% (w/v) Tween 20. In the case of Span 20, which has a lower HLB value, the mean diameters of nano-fibers were 271±68, 309±56 and 439±115 nm, respectively, at 1%, 2% and 4% (w/v) Span 20 (Table 1). There were significant decreases in the diameters of MAC nano-fibers including Tween 20 and Span 20, as compared with the polymer solution without surfactants. However, there were no significant differences in the diameters of MAC nano-fibers including Tween 20 at final concentrations between 1% and 4%. In contrast, the diameters of nano-fibers including 4% (w/v) Span 20 showed an approximately 1.6-fold increase as compared to that including 1% (w/v) Span 20. From these observations, it can be considered that the addition of non-ionic surfactants to the MAC polymer solution provides decreases in the surface tension of polymer solution and the diameters of nano-fibers formed. Moreover, in case of Span 20 but not Tween 20, it is considered that the formation of micelle in the MAC polymer solution plays a key role to increase diameter of nano-fibers because addition of surfactant above critical micelle concentration to polymer solution enhance a cross linking between polymer and surfactant.21)
As one application of MAC nano-fibers, we produced MAC nano-fiber tablets including a water-soluble model drug, UN. In our previous study, it was evidenced that UN embedded in the MAC nano-fibers are amorphous condition, which was determined by X-ray diffraction.13) Using a tablet machine, the MAC nano-fiber tablets could be shaped without lubricants such as magnesium stearate or talc, and it was found that there were no tableting faults such as capping, binding, lamination, picking or sticking. For in vitro drug release experiments, the amounts of Tween 20 or Span 20 in the MAC nano-fiber were set at 1%, 2% or 4% (w/w) to regulate sustained-release. In vitro drug release profiles of UN from the MAC nano-fiber tablets including Tween 20 or Span 20 are shown in Fig. 6.
Symbols with a bar represent the mean±S.D. of 6 experiments. Key: ■, Free; ◆, 1%; ▲, 2%; ●, 4%.
The percentage release of UN without surfactants (control) was about 80% up to 480 min. On the other hand, the percentage release of UN from tablets including Tween 20 at 1%, 2% and 4% (w/v) were about 92%, 100% and 100% up to 480 min, respectively (Fig. 6a). In contrast, as shown in Fig. 6b, the percentage release of UN from tablets including Span 20 at 1%, 2% and 4% (w/v) were approximately 87%, 90% and 90% up to 480 min, respectively, and there was no differences in the amount of UN released as compared with the control. These observations suggested that the addition of Tween 20 could regulate a water-soluble drug release from MAC nano-fiber tablets. Moreover, since the MAC nano-fiber tablets including an appropriate concentration Tween 20 can form a solid-in-oil-in-water (S/O/W) system after application to water phase, it also could enables us to regulate the release profiles of other water-soluble drugs.
In summary, the ES device we developed here could suppress fibers from spreading and prevent formation loss on the nano-fiber-collection plate. By attaching a stainless-steel conductor rod to the center of the nano-fiber-collection plate, nano-fibers could be collected more efficiently. The diameter of MAC nano-fibers was related to the permittivity of the solvent used for dissolving MAC and the concentration of MAC. For pharmaceutical applications, MAC nano-fiber tablets could be prepared without lubricants, and addition of Tween 20 into the tablets enabled regulation of the release profile of a water-soluble drug. Since nano-fibers of MAC is sub-nano material embedded drugs, by carrying out further appropriate bottom-down processing for nano-fibers, it is useful to manufacture sub-nano particles that can apply for injectable particles to perform tissue targeting or manufacturing core–shell type’s nano-capsules and so on.
The authors declare no conflict of interest.
