In recent years, advanced polymeric nanofibrous drug delivery systems (DDSs) have been of increasing use in pharmaceutical and biomedical applications. Such systems include oral dispersible thin films, ophthalmic preparations, multifunctional wound dressings, and implanted DDSs [1
]. These hybrid polymer nanofibers hold great promise with respect to drug therapy and tissue engineering in both human and veterinary medicine. Nanofibers have a unique capability to modify material properties, which could have a paramount effect on drug delivery performance. They possess fiber structure dimensions at the nanoscale and a large outer surface-to-volume ratio, enabling multiple alternatives in drug delivery. The conversion of polymer solutions to nanofibers by solution electrospinning (ES) is affected by material, process, and ambient parameters [3
]. The geometric and physicochemical properties of nanofibers depend, for example, on the nature of the solution, polymer type, polymer chain conformation, viscosity, elasticity, and electrical conductivity, as well as on solvent polarity and surface tension [4
]. These structural fiber properties in turn can have a great impact on the dissolution and drug release of fibers [6
]. To date, however, our understanding on the dissolution (drug release) behavior of polymeric nanofibers and related physicochemical mechanisms is still limited. This is partially due to the fact that the nanofibers are challenging samples for analysis due to their nanoscale size and brittleness.
In the state-of-the art literature there are many examples of how the geometric nanofiber properties (size, size distribution, surface morphology, orientation) and porosity can greatly affect the dissolution (drug release) and therapeutic efficiency of nanofibrous DDSs [6
]. With poorly water-soluble drugs, the limitations in drug release of the nanofibrous DDSs can delay the onset of drug action, cause poor oral bioavailability, and ultimately decrease therapeutic effect. The geometric nanofiber properties can be governed by the adjustment and control of the critical material, process, and environmental parameters in an ES process [5
]. Lately, increasing interest has been focused on developing ES processes which could produce drug-loaded nanofibers of a specific quality and with reproducible drug release. Due to the nanoscale size, controlling the quality and performance of such nanofibers is still challenging. Consequently, the mechanisms of drug release associated with such novel polymeric nanofibrous DDSs remain poorly understood.
Scanning white light microscopic interferometry (SWLI) is a high-depth resolution imaging technique suitable for determining the geometric properties (i.e., fiber shape, diameter, and length), porosity, and surface topography of nanofibrous DDSs [17
]. Three-dimensional (3D) SWLI permits rapid, non-contacting and non-destructive imaging of nanofibrous samples, since it requires neither sample preparation nor modification. Recently, we applied SWLI and high-resolution scanning electron microscopy (SEM) equipped with a customized measurement program for 3D surface topography analysis of non-woven nanofibrous mats [17
]. In pharmaceutical research, the use of SWLI for investigating and non-contact imaging of the manufacturing processes and final products is still limited. Sandler et al. and Genina et al. used SWLI to image 3D printed multi-layered DDSs [18
]. Hanhijärvi et al. applied SWLI to investigate the film surface properties of hydroxypropyl methylcellulose (HPMC)-coated tablets [20
], while O’Bryan et al. used it to image 3D printed microgels intended for bioprinting applications [21
]. Recently, Wickström et al. applied SWLI to characterize the surface texture and surface roughness of antibiotic-loaded fiber-reinforced composite implants [22
The aim of the present study was to provide insight into the wetting and early-stage dissolution of drug loaded polymeric nanofibers. To gain an understanding of such phenomena, we monitored in situ the wetting and drug release of fibers loaded with a poorly water-soluble drug (piroxicam, PRX), and determined the solid-state of the drug during dissolution. Two hydrophilic polymers were investigated in nanofibers: hydroxypropyl methylcellulose (HPMC) intended for the sustained drug release and polydextrose (PD) intended for the immediate drug release of a poorly water-soluble drug [23
]. We combined SWLI and high-resolution optical microscopy to image PRX-loaded nanofibers in contact with an aqueous medium, and the physical appearance and solid-state changes of nanofibers were verified with scanning electron microscopy (SEM), Raman spectroscopy, and X-ray diffraction (XRD), respectively. To our knowledge, this is the first time when such imaging and solid-state analyses are applied in a complimentary fashion to investigate the dissolution of polymeric nanofibers.
2. Materials and Methods
Commercially available cellulose ether, hydroxypropyl methylcellulose, HPMC (Methocel K100M premium CR, Colorcon Ltd., Dartford, Kent, UK), and polydextrose, PD (STA-LITE L90, 70% aqueous solution of PD, Tate & Lyle Netherlands B.V., Koog Aan De Zaan, KA, Netherlands) (Figure 1
) were used as carrier polymers for the ES of nanofibers. The organic solvents were 1,1,1,3,3,3-hexa-fluoro-2-propanol (HFIP) (≥99.0%) (Sigma-Aldrich C.C., St. Louis, MO, USA) and methanol (Lach-Ner, s.r.o., Neratovice, Czech Republic). Anhydrous piroxicam, (PRX, pure form I, PRXAH I, Letco Medical, Inc., N Livonia, MI, USA) was selected as a model drug for a low-dose poorly water-soluble active substance in nanofibers. In the wetting and dissolution experiments, purified water, hydrochloric acid buffer solution, USP 28 (pH 1.2), and phosphate buffer solution (pH 7.2) were used as dissolution media. The materials for the preparation of buffer solutions were of analytical grade and purchased from Lach-Ner, s.r.o., Neratovice, Czech Republic.
2.2. Preparation of Fiber Mats
The carrier polymer (HPMC) and model drug (PRX) were first manually dry mixed with a mortar and pestle at three different ratios (1:1, 1:2, and 1:4). Next, the pre-mixtures were dissolved in organic solvent to obtain the 0.8% (w
) solution of HPMC in HFIP as described in our previous study [16
]. With PD, the aqueous solution of PD (70%) alone or the mixture of the aqueous solution of PD (70%) and methanol (5:1) were used for ES. The amount of PRX in the 70% aqueous solution of PD (0.75 mL) used for ES was 20 mg, and 0.75 mL contained 530 mg PD. The drug-loaded nanofibers were fabricated with an in-house ES set-up equipped with a syringe system, automatic syringe pump, spinneret, high-voltage power supply, and a grounded collector plate. The high-voltage power supply system was a Gamma (Gamma High Voltage Research, Model ES3OP-10W/DAM, Ormond Beach, FL, USA). For HPMC solutions, the ES distance and voltage between the spinneret and the collector plate were 8.0 cm and 7 kV, respectively. The pump rate of the automatic syringe pump (KdScientific, Model No: KDS-250-CE, Geneq Inc, Montreal, Quebec, Canada) was 1.0 mL/h. For PD solutions, the ES distance, voltage, and pump rate were 17 cm, 14 kV, and 2.0 mL/h, respectively. The prepared fibers were stored in desiccators (above silica gel, with relative humidity (RH) of 0%) at ambient room temperature (21 ± 2 °C) and in a refrigerator (6 ± 2 °C/RH 0%) prior to use.
2.3. Physical Appearance and Solid-State Characterization
The size and surface morphology of fibers were investigated with scanning electron microscopy, SEM (Helios NanoLab 600, FEI Company, Hillsboro, OR, USA). The samples were coated with a 3-nm gold layer in an argon atmosphere using a magnetron sputter prior to SEM imaging. Solid-state characterization of the starting materials and the electrospun fibers were carried out with X-ray diffraction, XRD (D8 Advance, Bruker AXS GmbH, Karlsruhe, Germany), and Raman spectroscopy (B&W Tek Inc., Newark, DE, USA). In XRD, Cu Kα1 radiation (λ= 1.5418 Å, 40 kV, and 40 mA) was used, and data were collected in the range of 5° and 35° 2θ with a step size of 0.2° 2θ. The brittleness of the fibrous mats was detected by visual inspection and manual palpation with tweezers.
2.4. In Situ Wetting and Dissolution Tests of Fibers
For in-situ wetting tests, we applied the fiber mats as such and the tablets compressed from these fibers. The initial-stage dissolution of the fiber mats were monitored by a high-resolution optical microscopy (Microscope Leica DMLB equipped with the Leica Germany 5.0× and L50×/0.50 objectives and Canon Power Shot 550 digital camera). A standardized drop (50 μL) of aqueous dissolution media was gently placed in contact with a fiber mat at room temperature (21 ± 2 °C), and the dissolution was monitored by taking micrographs at regular intervals.
Pieces of a fiber mat were manually compressed into small flat-faced tablets (discs) with a miniaturized press (Specac Ltd., Orpington BR5 3FQ, UK) intended for preparing sample discs for infrared (IR) spectroscopy analysis. The compression pressure was 196 MPa (2t/cm2) and the compression time was 5 s. The tablets (discs) were 12.7 mm in diameter and 0.8–1.0 mm in thickness. For in situ initial-stage dissolution experiments, one micro-pipette drop of purified water (2 µL) was gently placed onto the surface of fibrous tablets at room temperature (21 ± 2 °C). The contact point of the drop onto the tablet surface was kept as constant as possible.
The wetting and early-stage dissolution of fibrous tablets were monitored in situ with an in-house 3D scanning white light microscopic interferometry (SWLI). In SWLI, a light beam passes through an interferometric objective (Nikon, Michelson type, magnification 120×) containing a beam splitter that reflects half the incident beam onto a reference surface and passes the other half onto the test surface (Figure 2
). Light reflected from the test and reference surfaces recombines and interferes to form an interferogram. The working principle and structure of SWLI is described elsewhere. For details see Kassamakov et al. [25
], Hanhijärvi et al. [20
], and Paaver et al. [17
]. In the present study, we obtained three-dimensional (3D) images featuring 29 nm × 29 nm active pixel size from a 55 × 40 μm2
area. The time period of 10 min prior to a subsequent drop was required for the complete SWLI analysis of the tablets. Purified water (pH 5.6) and the USP 28 buffer solutions (hydrochloric acid buffer pH 1.2 and phosphate buffer solution pH 7.2) were used as dissolution media. The total number of optical microscopy and SWLI images taken on the course of the in situ dissolution experiments was over 500 (including short video-clips). The number of samples was 3–6 in each analysis.
A high-resolution scanning electron microscope (SEM) (Helios NanoLab 600, FEI Company, Hillsboro, OR, USA) was used as a reference method to the SWLI. A software-based measurement function xT Microscope Control (FEI) was used to measure the horizontal dimensions of the nanofibers. Samples were mounted on aluminum stubs with silver paint and were magnetron-sputter coated with a 3-nm gold layer in an argon atmosphere prior to microscopy. ImageJ (vers. 1.50i) was used for fiber size distribution analysis.
2.5. Dissolution Test
The in vitro dissolution tests of the electrospun nanofiber mats and the pure PRX powder samples were performed using an USP dissolution apparatus I (basket method) in a semi-automated dissolution system (Termostat-Sotax AT7, Sotax AG, Aesch, Switzerland). The concentration of PRX in the dissolution medium was measured at 354 nm by using a UV-VIS spectrophotometer (Ultrospec III, Biochrom Ltd, Cambourne, Cambridge, UK). The dissolution medium was 900 mL of purified water (pH 5.6) and buffer solution (pH 1.2 or pH 7.2) maintained at 37 ± 0.5 °C as described in the USP 28. The basket rotation speed was 50 rpm. The amount of PRX in the powder and nanofiber samples used in the dissolution test was 20 mg. Six parallel tests were performed for each sample.