Hydroxypropyl-β-Cyclodextrin inclusion complex nanofibrous webs as fast-dissolving oral drug delivery system

Journal Pre-proofs Metronidazole/Hydroxypropyl-β-Cyclodextrin Inclusion Complex Nanofibrous Webs as Fast-dissolving Oral Drug Delivery System Asli Celebioglu, Tamer Uyar PII: DOI: Reference:

S0378-5173(19)30873-7 https://doi.org/10.1016/j.ijpharm.2019.118828 IJP 118828

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

14 August 2019 21 October 2019 26 October 2019

Please cite this article as: A. Celebioglu, T. Uyar, Metronidazole/Hydroxypropyl-β-Cyclodextrin Inclusion Complex Nanofibrous Webs as Fast-dissolving Oral Drug Delivery System, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/j.ijpharm.2019.118828

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Metronidazole/Hydroxypropyl--Cyclodextrin Inclusion Complex Nanofibrous Webs as Fast-dissolving Oral Drug Delivery System Asli Celebioglu* and Tamer Uyar* Department of Fiber Science & Apparel Design, College of Human Ecology, Cornell University, Ithaca, NY, 14853, United States *Corresponding

Authors: AC: [email protected]; TU: [email protected]

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ABSTRACT: In this study, Metronidazole/Hydroxypropyl--Cyclodextrin (HP-β-CyD) inclusion complex nanofibrous webs were produced via electrospinning for the purpose of fast-dissolving oral drug delivery. The Metronidazole/HP-β-CyD inclusion complex aqueous solutions having two different molar ratio of Metronidazole/HP-β-CyD (1/1 and 1/2) were prepared by using very high concentration of HP-β-CyD (200 %, w/v) in order to achieve polymer-free electrospinning of Metronidazole/HP-β-CyD nanofibers (NF). Metronidazole was totally encapsulated and preserved without any loss during the electrospinning process in which both systems yielded Metronidazole/HP-β-CyD NF having the same initial molar ratio of 1/1 and 1/2. The electrospinning of both Metronidazole/HP-β-CyD (1/1 and 1/2) aqueous solutions yielded uniform and bead-less fiber morphology having ~190 nm average fiber diameter. Both Metronidazole/HP-β-CyD NF (1/1 and 1/2) samples were in the form of nanofibrous webs with free-standing and flexible character. The structural and thermal characterizations of Metronidazole/HP-β-CyD NF (1/1 and 1/2) proved that Metronidazole was in the inclusion complex state with HP-β-CyD in these nanofibrous webs. Metronidazole is an antibiotic which is poorly water-soluble drug, but the phase solubility and dissolution tests revealed that the watersolubility of Metronidazole was significantly enhanced by HP-β-CyD inclusion complexation. In addition, Metronidazole/HP-β-CyD (1/1 and 1/2) nanofibrous webs have shown very fastdissolving behavior when placed in water or contacted to artificial saliva suggesting that such Metronidazole/HP-β-CyD nanofibrous webs can be suitable for fast-dissolving oral drug delivery. Keywords: metronidazole; hydroxypropyl-beta-cyclodextrin; electrospinning; nanofibers; fastdissolving; oral drug delivery

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1. Introduction The development of fast-dissolving drug delivery systems is on the rise in the pharmaceutical industry (Bala et al., 2013; Chowdary et al., 2012; Kathpalia and Gupte, 2013; Liang and Chen, 2001; Nagaraju et al., 2013). A fast-dissolving oral drug delivery system readily dissolves and/or disintegrates in the oral cavity without the need of water or chewing, therefore, it is very convenient for consumers. Moreover, the fast-dissolving drug delivery system enables the fast absorption of drug and improves its bioavailability. Therefore, fast-dissolving oral drug delivery system is very promising way of drug administration for everyone, specifically for patients with swallowing difficulties as well as for pediatric and geriatric patients (Bala et al., 2013; Chowdary et al., 2012; Kathpalia and Gupte, 2013; Liang and Chen, 2001; Nagaraju et al., 2013). Fastdissolving oral drug delivery systems are mostly in tablet form (Parkash et al., 2011; Rahane and Rachh, 2018) or they can be in film/strip form as well (Bala et al., 2013; Nagaraju et al., 2013). Very recently, there is an interest in developing fast-dissolving drug delivery systems from nanofibers by using electrospinning technique (Seif et al., 2015; Yu et al., 2018). The solution electrospinning technique is a quite versatile for the incorporation of drug molecules in the fiber matrix where the common solution mixture of biopolymer matrix and drug is electrospun to produce nanofibers made of drug/biopolymer composition in the form of nanofibrous mats (Seif et al., 2015; Uyar and Kny, 2017; Yu et al., 2018). The rapid solvent evaporation during the electrospinning process prevents the drug crystallization, and solid dispersion of drug molecules in amorphous state within the fiber matrix is produced (Nagy et al., 2015; Seif et al., 2015; Yu et al., 2018). The free-standing electrospun nanofibrous webs/mats/membranes incorporating drugs have very large surface area and highly porous structure, and the drug molecules are mostly in amorphous state, therefore, such nanofibrous webs would readily dissolve with water contact.

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Hence, electrospun nanofibers and their nanofibrous webs can be promising candidates for developing fast-dissolving oral drug delivery systems (Yu et al., 2018). For the fast-dissolving drug delivery systems, the choice of polymeric matrix for the electrospinning is water-soluble biopolymers such gelatin (Aytac et al., 2019; Kwak et al., 2017; Mano et al., 2017), polyvinylpyrrolidone (PVP) (Bukhary et al., 2018; Illangakoon et al., 2014; Quan et al., 2011; Yu et al., 2009), poly(vinyl alcohol) (PVA) (Li et al., 2013; Manasco et al., 2014; Nam et al., 2017), chitosan/pullulan (Qin et al., 2019), etc. Variety of drug molecules including ciprofloxacin (Aytac et al., 2019), caffeine (Illangakoon et al., 2014), ibuprofen(Yu et al., 2009), feruloyl-oleyl-glycerol (Quan et al., 2011), paracetamol (Illangakoon et al., 2014), ketoprofen (Manasco et al., 2014), riboflavin (Li et al., 2013), aspirin (Qin et al., 2019), etc. were incorporated into electrospun nanofibers and these drug/biopolymer nanofibrous materials have shown fast-dissolving behavior. Very recently, we have shown that electrospun nanofibrous webs based on cyclodextrin inclusion complexes of drugs (eg. sulfisoxazole (Yildiz et al., 2017), paracetamol (Yildiz and Uyar, 2019), ibuprofen (Celebioglu and Uyar, 2019) without using any polymers can be suitable for fast-dissolving oral drug delivery systems. The polymer-free electrospun nanofibers of cyclodextrin inclusion complexes of drugs (eg., spironolactone (Vigh et al., 2013), diclofenac sodium (Balogh et al., 2015) , voriconazole (Vass et al., 2019)) having fast-dissolving behavior were also reported by other research groups. The use of cyclodextrins as a fiber matrix has certain advantages over polymeric matrix since cyclodextrins form inclusion complexation with drug molecules and make the drug molecules water-soluble and such inclusion complexation also increase the stability and the bioavailability of the drugs (Bilensoy, 2011; Carneiro et al., 2019). Cyclodextrins are cyclic oligosaccharides which are used in pharmaceutics since they

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increase the stability, water solubility and bioavailability of the drug molecules, as well as they provide controlled/sustained delivery of the drug molecules (Bilensoy, 2011). Several studies related to electrospinning of nanofibers from biopolymers incorporating cyclodextrin inclusion complexes of drugs were reported for the purpose of sustained/controlled release of drugs (Aytac et al., 2015; Canbolat et al., 2014; Monteiro et al., 2017; Topuz and Uyar, 2019). Moreover, fastdissolving drug delivery systems based on electrospun nanofibers of biopolymers incorporating drug/cyclodextrin inclusion complexes is also possible (Aytac et al., 2019; Manasco et al., 2014; Samprasit et al., 2018). Yet, the polymer-free electrospun nanofibers of drug/cyclodextrin inclusion complexes have ultra-fast dissolving behavior (Balogh et al., 2015; Celebioglu and Uyar, 2019; Topuz and Uyar, 2019; Vass et al., 2019; Vigh et al., 2013; Yildiz et al., 2017; Yıldız and Uyar, 2019) compared to polymeric nanofibrous systems (Aytac et al., 2019; Manasco et al., 2014; Topuz and Uyar, 2019). Metronidazole is an antibiotic that is used to treat a wide variety of infections, but it is a poorly water-soluble hydrophobic drug. Metronidazole is quite effective for infectious diseases of oral cavity and periodontitis (Flemmig et al., 1998; Perioli et al., 2004). Therefore, there are different approaches reported in the literature in which the efficacy of Metronidazole was improved using different carrier systems for the treatment of buccal cavity (Bottino et al., 2014; Jones et al., 2008; Perioli et al., 2004; Reise et al., 2012). Here, we have chosen Metronidazole as poorly water-soluble model drug molecule for the development of fast-dissolving oral drug delivery

system

based

on

polymer-free

electrospun

nanofibrous

webs

of

Metronidazole/cyclodextrin inclusion complex. It has been shown that cyclodextrins can form inclusion complex with Metronidazole for controlled release (Stjern et al., 2017), and for improved stability and water solubility (Andersen and Bundgaard, 1984). Studies related to 5

encapsulation of Metronidazole within electrospun nanofibers for its controlled delivery for the purposes of tissue engineering, dental and other biomedical applications have been also reported (Bottino et al., 2014, 2013; He et al., 2017, 2015, 2018; Kamocki et al., 2015; Palasuk et al., 2014; Passos et al., 2019; Reise et al., 2012; Schkarpetkin et al., 2016; Wang et al., 2019; Xue et al., 2015b, 2015a, 2014c, 2014a, 2014b; Zupančič et al., 2018, 2016). Yet, to the best of our knowledge, there is no study related to fast-dissolving electrospun nanofibrous webs incorporating Metronidazole. The present study summarizes our effort to develop fast-dissolving oral drug delivery system based on polymer-free electrospun nanofibrous webs of Metronidazole/cyclodextrin inclusion complex. 2. Materials and methods 2.1. Materials Hydroxypropyl-beta-cyclodextrin (HP-β-CyD) was provided by Wacker Chemie AG (USA) as a free-sample. Metronidazole (analytical standard, Sigma Aldrich), potassium phosphate monobasic (KH2PO4, ≥99.0%, Fisher Chemical), sodium phosphate dibasic heptahydrate (Na2HPO4, 98.0-102.0%, Fisher Chemical), sodium chloride (NaCl, >99%, Sigma Aldrich), ophosphoric acid (85% (HPLC), Fisher Chemical) and deuterated dimethylsulfoxide (d6-DMSO, 99.8%, Cambridge Isotope) were purchased and used without further purification. The highquality distilled water was sourced of the Millipore Milli-Q ultrapure water system. 2.2. Electrospinning process Firstly, the hydroxypropyl-beta-cyclodextrin (HP-β-CyD) was dissolved in distilled water by 200% (w/v) solid concentration. Then, Metronidazole powder was mixed in the clear solutions of HP-β-CyD to get 1/1 and 1/2 Metronidazole/HP-β-CyD molar ratios, separately. The inclusion complexation between Metronidazole and HP-β-CyD was guaranteed by 24 hours stirring at 6

room temperature. While, the solution of Metronidazole/HP-β-CyD (1/1) became turbid, Metronidazole/HP-β-CyD (1/2) system became clear by the end of 24 h mixing. For comparative study, the pure HP-β-CyD solution was also prepared and electrospun into homogenous nanofibers by 200% (w/v) HP-β-CyD (w/v) concentration. The solution of Metronidazole/HP-βCyD (1/1), Metronidazole/HP-β-CyD (1/2) and HP-β-CyD was filled in 1 mL syringe fixed with 27 G metal needle and filled syringes were placed onto the syringe pump (New Era, USA), separately. The flow rate of syringe pump was arranged to 0.5 mL/h and the stable voltage of 15 kV was ensured by high voltage power supply (EPR series, Matsusada, Japan) during the electrospinning process. The grounded metal collector, which was wrapped with a piece of aluminium foil, was located at a distance of 15 cm from the tip of the needle and used for the deposition of nanofibrous webs. The electrospinning process was carried out in a Plexiglass box, and the ambient temperature and humidity were recorded as 21 oC and 55%, respectively. The electrospun nanofibers produced from the solutions of pure HP-β-CyD, Metronidazole/HP-βCyD inclusion complexes with 1/1 and 1/2 molar ratio were emitted as HP-β-CyD NF, Metronidazole/HP-β-CyD (1/1) NF and Metronidazole/HP-β-CyD (1/2) NF, respectively. 2.3. Solution properties The viscosity of HP-β-CyD solution and Metronidazole/HP-β-CyD solutions (1/1 and 1/2, molar ratio) was determined by rheometer (AR 2000 rheometer, TA Instrument, USA) equipped with 20 mm cone/plate accessory (CP 20-4 spindle type, 4o). The process distance was set to 108 µm and the measurements were recorded at the range of 0.01-1000 s-1 shear rate and at 22 oC. The conductivity-meter (FiveEasy, Mettler Toledo, USA) was used to check the conductivity of the same solutions.

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2.4. Morphological characterization The morphology of HP-β-CyD NF, Metronidazole/HP-β-CyD (1/1) NF and Metronidazole/HPβ-CyD (1/2) NF was examined using scanning electron microscope (SEM, Tescan MIRA3, Czech Republic). The samples were sputtered with a thin layer of Au/Pd to eliminate charging in the course of SEM imaging. The accelerating voltage and the working distance were arranged to 12 kV and 10 mm, respectively. ImageJ software was run to calculate the average fiber diameter (AFD) of nanofibers from different locations of SEM images (~100 fibers). 2.5. Proton nuclear magnetic resonance spectroscopy The molar ratio between Metronidazole and HP-β-CyD in Metronidazole/HP-β-CyD NF was calculated by proton nuclear magnetic resonance (1H-NMR) spectrometer (Bruker AV500, with autosampler). The 1H-NMR solutions were prepared by dissolving Metronidazole powder, HP-βCyD NF, Metronidazole/HP-β-CyD (1/1) NF and Metronidazole/HP-β-CyD (1/2) NF in d6DMSO at the 40 g/L sample concentration, separately. The 1H-NMR spectra were recorded upon 16 scanning and Mestranova software was used to integrate the chemical shifts (δ, ppm) of each sample. The integration of peaks of Metronidazole (-N-CH2; 4.3ppm) and HP-β-CyD (-CH3; 1.03 ppm) were used to calculate the molar ratio of Metronidazole/HP-β-CyD (1/1) NF and Metronidazole/HP-β-CyD (1/2) NF. 2.6. The Fourier transform infrared spectroscopy The Fourier transform infrared (FT-IR) spectra of Metronidazole powder, HP-β-CyD NF, Metronidazole/HP-β-CyD (1/1) NF and Metronidazole/HP-β-CyD (1/2) NF were recorded using Attenuated total reflectance Fourier transform infrared (ATR-FT-IR) spectrometer (PerkinElmer, USA). The measurements were performed between 4000 and 600 cm−1 at a resolution of 4 cm−1 and upon 64 scans. 8

2.7. Thermal analyses Differential scanning calorimeter (DSC, Q2000, TA Instruments, USA) and thermogravimetric analyzer (TGA, Q500, TA Instruments, USA) were used to examine the thermal characteristics of samples. For DSC measurements, samples were sealed into Tzero aluminum pan and heated from 0 oC to 240 oC under N2 atmosphere upon 10 oC/min heating rate. Prior the TGA measurements, samples were balanced onto platinum TGA pan and heated from room temperature to 550 oC at a heating rate of 20 °C/min under N2 atmosphere during the measurements. 2.8. X-ray diffraction X-ray diffraction (XRD) patterns of Metronidazole powder, HP-β-CyD NF, Metronidazole/HPβ-CyD (1/1) NF and Metronidazole/HP-β-CyD (1/2) NF were investigated by X-ray diffractometer (Bruker D8 Advance ECO). Prior the measurement, the voltage and current were set to 40 kV and 25 mA, respectively. The XRD patterns were scanned between the 2ϴ angles of 5o and 30o by applying Cu-Kα radiation. 2.9. Phase solubility analysis Phase solubility profile of Metronidazole in HP-β-CyD solutions was examined according to method reported by Higuchi and Conners (Higuchi and Connors, 1965). A known amount of Metronidazole above its solubility and HP-β-CyD powder with an increasing concentration (0120 mM) were weighted into glass vials, separately. Afterwards, 5 mL of water was added each of the vial and the vials were sealed. The sealed vials were shielded from the light and shaken for 24 h on incubator shaker at 25 oC and 450 rpm. After 24 hours equilibrium, the suspensions were filtered with 0.45µm PTFE filter. UV-Vis-spectroscopy (PerkinElmer, Lambda 35) was used to calculate the amount of Metronidazole dissolved in the filtered aliquots by taking account the 9

absorption value at 319 nm. The experiments were carried out in triplicate (n=3), the phase solubility diagram was plotted from the average results and according to calibration curve which showed linearity and acceptability with R2≥0.99 (Fig. S1). The binding constant (Ks) was calculated, as well from the following equation;

Ks =

slope S0 (1 ― slope)

where S0 is the intrinsic solubility (~ 54mM) (Malli et al., 2018) of Metronidazole in the absence of HP-β-CyD. 2.10. In vitro dissolution tests The time dependent dissolution profiles of Metronidazole in Metronidazole/HP-β-CyD (1/1) NF and Metronidazole/HP-β-CyD (1/2) NF webs at the same weight of ~ 40 mg was investigated in distilled water (50 mL) at room temperature. For comparison, the time dependent dissolution profile of ~ 4 mg of Metronidazole powder was also examined which corresponds to theoretical Metronidazole content in Metronidazole/HP-β-CyD (1/1) NF. For the test, the nanofibrous webs and Metronidazole powder were precisely weighed and placed into a beaker, then, water medium was poured into samples. The solutions were shaken with the speed of 200 rpm and 0.5 mL of sample solution was withdrawn and an equal amount of fresh medium was refilled at the given time points. The dissolved amount of Metronidazole was analyzed using UV-Vis-spectroscopy at the wavelength of 319 nm. The calibration curve of Metronidazole showed linearity and acceptability with R2≥0.99, and measurement results were adapted to this calibration curve to convert absorbance to concentration (µg/mL). The experiments were performed three times and results were reported as average.

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2.11. Solubility, dissolution and disintegration profile of Metronidazole/HP-β-CyD NF webs The solubility enhancement of Metronidazole encapsulated in Metronidazole/HP-β-CyD NF was also demonstrated after a long term stirring of Metronidazole powder and Metronidazole/HP-βCyD NF solutions (24 hours). For this experiment, the solution of Metronidazole/HP-β-CyD (1/1) NF and Metronidazole/HP-β-CyD (1/2) NF were prepared with the web concentration of ~ 0.3 g/mL. The Metronidazole powder concentration was kept at ~100 mM which corresponds to the equivalent amount of Metronidazole in Metronidazole/HP-β-CyD (1/2) NF web. All systems were stirred in distilled water for 24 h at 150 rpm. The undissolved part of Metronidazole in the solutions was removed by 0.45 µm PTFE filter and the UV-Vis measurements were performed in the range of 200-450 nm. For the dissolution test, Metronidazole (~2 mg), Metronidazole/HP-β-CyD (1/1) (~19 mg) and Metronidazole/HP-β-CyD (1/2) (~35 mg) NF web having the equivalent Metronidazole content were weighted into glass vials. For comparison, the pristine HP-β-CyD NF web (~35 mg) were also weighted and placed into glass vial. In order to monitor the dissolution, a video was recorded synchronously with the addition of distilled water (3 mL) into vials. The disintegration profiles of Metronidazole/HP-β-CyD NF webs were examined with the modified version of a method that was developed by. Bi et al. at which the physiological conditions under the surface of a moist tongue were simulated (Bi et al., 1996). In this method, a filter papers having proper size were located in plastic petri dishes (10 cm), and then they were wetted with 10 mL of artificial saliva (The aqueous mixture of 16.8 mM Na2HPO4, 1.4 mM KH2PO4 and 137 mM NaCl were prepared and pH was arranged as 6.8 by the addition of phosphoric acid). Then, the excess artificial saliva was drained away from the petri dishes and a

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piece of Metronidazole/HP-β-CyD NF webs were put at the center of the filter paper. The disintegration of Metronidazole/HP-β-CyD NF webs was recorded as video. 3. Results and discussions 3.1. Morphological characterization Cyclodextrins (CyDs) make hydrophobic drug molecules water-soluble by forming inclusion complexation (Brewster and Loftsson, 2007), therefore, the electrospinning of drug/cyclodextrin systems can be performed in water without the need of using toxic organic solvents (Vass et al., 2019; Yildiz et al., 2017; Yıldız and Uyar, 2019). On the contrary, the electrospinning of drug/polymer systems often requires organic solvents or organic solvent/water mixtures in order to dissolve both polymeric matrix and hydrophobic drugs (Giram et al., 2018; Nagy et al., 2015; Yu et al., 2018). In this study, we have performed the electrospinning of Metronidazole/HP-βCyD nanofibers (NF) from aqueous solution of Metronidazole/HP-β-CyD inclusion complex (Fig. 1). Since very high concentration of CyD solution is needed for electrospinning of uniform nanofibers (Celebioglu and Uyar, 2012), we optimized the HP-β-CyD concentration as 200% (w/v) which resulted in free-standing nanofibrous web having defect-free and bead-less uniform fiber morphology (Fig. 2a-i,ii). Then, we prepared 1/1 molar ratio of Metronidazole/HP-β-CyD aqueous solution by keeping the concentration of HP-β-CyD as 200% (w/v). Most of the Metronidazole powder became soluble in Metronidazole/HP-β-CyD (1/1), yet, the solution was somewhat turbid indicating the presence of some undissolved Metronidazole crystals (Fig. 2b-ii). By decreasing the content of Metronidazole to half, we obtained clear solution for Metronidazole/HP-β-CyD (1/2) confirming that Metronidazole powder was totally dissolved and become water-soluble by inclusion complexation with HP-β-CyD (Fig. 2c-ii).

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The electrospinning of both Metronidazole/HP-β-CyD (1/1) and Metronidazole/HP-βCyD (1/2) aqueous solutions yielded in bead-less and uniform fiber morphology (Fig. 2). The average fiber diameter (AFD) of pristine HP-β-CyD NF, Metronidazole/HP-β-CyD (1/1) NF and Metronidazole/HP-β-CyD (1/2) NF were calculated as 220±60 nm, 190±100 nm and 195±80 nm, respectively (Table 1). The AFD of Metronidazole/HP-β-CyD (1/1) NF and Metronidazole/HPβ-CyD (1/2) NF was almost same since viscosity and conductivity values of both solutions were very close to each other. The AFD of pristine HP-β-CyD NF was a bit higher due to higher viscosity of the HP-β-CyD solution. The addition of Metronidazole to HP-β-CyD solution lower the viscosity of the solution but did not change much the conductivity of the solution, only a minor decrease in solution conductivity was recorded. The solution properties such as viscosity and conductivity are quite important which they play a major role in electrospinning process resulting different fiber morphology and fiber diameters (Uyar and Besenbacher, 2008). Here, the incorporation of different amount of Metronidazole (1/1 and 1/2) in HP-β-CyD solution had only slight change for the viscosity and conductivity of the HP-β-CyD solution, hence, all the electrospun samples of pristine HP-β-CyD NF, Metronidazole/HP-β-CyD (1/1) NF and Metronidazole/HP-β-CyD (1/2) NF yielded uniform fiber morphology having similar fiber diameter

(AFD

of

~190

nm).

Moreover,

Metronidazole/HP-β-CyD

(1/1)

NF

and

Metronidazole/HP-β-CyD (1/2) NF samples were in the form of self-standing and flexible webs which make them applicable for oral drug delivery as fast-dissolving systems. 3.2. Structural characterization The Metronidazole/HP-β-CyD aqueous solutions have initial molar ratio of 1/1 and 1/2 for the electrospinning which corresponds to the ~ 10 % (w/w, with respect to total sample amount) and ~ 6 % (w/w, with respect to total sample amount) of Metronidazole in nanofiber matrix, 13

respectively. However, the molar ratio of Metronidazole/HP-β-CyD may change for the nanofibers of Metronidazole/HP-β-CyD after the electrospinning process. Therefore, we performed 1H-NMR analyses determine the encapsulation efficiency and the final molar ratio of Metronidazole/HP-β-CyD in electrospun Metronidazole/HP-β-CyD (1/1 and 1/2) NF samples. The molar ratio of Metronidazole/HP-β-CyD was determined by calculating the proportion of the integrated peaks specific to Metronidazole and HP-β-CyD. The -CH3 protons of HP-β-CyD at 1.03 ppm and -N-CH2 protons of Metronidazole at 4.3 ppm were used for the molar ratio calculation of Metronidazole/HP-β-CyD in electrospun Metronidazole/HP-β-CyD (1/1 and 1/2) NF samples (Fig. 3). The 1H-NMR analyses showed that the initial molar ratio of 1/1 and 1/2 was preserved for Metronidazole/HP-β-CyD (1/1) NF and Metronidazole/HP-β-CyD (1/2) NF. It is important to achieve high yield of drug encapsulation for the electrospun Metronidazole/HP-βCyD NF samples. 1H-NMR results confirmed that Metronidazole was completely preserved during the electrospinning process by the loading capacity of ~ 10 % (w/w) and ~ 6 % (w/w) for Metronidazole/HP-β-CyD (1/1) NF and Metronidazole/HP-β-CyD (1/2) NF, respectively. In brief, Metronidazole/HP-β-CyD NF were produced without loss of Metronidazole keeping the initial molar ratio of 1/1 and 1/2 (Metronidazole/HP-β-CyD) and so the encapsulation efficiency of ~100 % was successfully achieved. Even though,

1H-NMR

is a powerful technique to determine the presence of

Metronidazole and molar ratio of Metronidazole/HP-β-CyD in electrospun Metronidazole/HP-βCyD (1/1 and 1/2) NF samples, the presence of Metronidazole in Metronidazole/HP-β-CyD (1/1 and 1/2) NF samples was also proved using FT-IR technique. The FT-IR spectra of pure Metronidazole powder, pristine HP-β-CyD NF and Metronidazole/HP-β-CyD (1/1 and 1/2) NF samples were given in Fig. 4. Pristine HP-β-CyD NF sample has prominent characteristics peaks 14

at 1028, 1150 and 1180 cm-1 due to the vibrations of coupled C–C/C–O stretching and antisymmetric C–O–C glycosidic bridge stretching. The other absorption bands at 3000-3600, 2930, 1650 and 1370 cm-1 are due to the primary /secondary -OH stretching, C-H stretching, OH bending and -CH3 bending vibrations of HP-β-CyD, respectively (Aytac et al., 2019; Yıldız and Uyar, 2019; Yuan et al., 2015) (Fig. 4a). For Metronidazole, the absorption bands due to NO stretching (1534 cm-1), O-H deformation (1426 cm-1), NO2 symmetric stretching (1368 cm-1), O-H in plane deformation (1263 cm-1), C-O stretching (1186 cm-1) (Fig. 4b), C–N stretching (823 cm-1) and (CH2)2 rocking vibration (743 cm-1) (Fig. 4c) were also observed in the FT-IR spectra of Metronidazole/HP-β-CyD (1/1 and 1/2) NF confirming the presence of Metronidazole in these samples (Ramukutty and Ramachandran, 2012). The XRD data of Metronidazole powder, pristine HP-β-CyD NF and Metronidazole/HPβ-CyD (1/1 and 1/2) NF samples are given in Fig. 5a. Metronidazole is a crystalline drug, which has characteristic diffractions at 12.2° and 13.8° (He et al., 2017). HP-β-CyD is an amorphous CyD type, so, the electrospun pristine HP-β-CyD NF is also amorphous having a broad XRD pattern without any sharp peaks. The XRD pattern of Metronidazole/HP-β-CyD (1/1) NF is very similar to that of pristine HP-β-CyD NF, but, the diffraction peak of Metronidazole crystals at 12.2° was observed in a very low intensity suggesting that very few amounts of Metronidazole crystals were present in this sample (Fig. 5a). The XRD data correlates with the visual observation of Metronidazole/HP-β-CyD (1/1) solution, which was turbid because of the some uncomplexed/undissolved Metronidazole crystals (Fig. 2b). On the other hand, there was no characteristic diffraction peak observed for Metronidazole from the XRD pattern of Metronidazole/HP-β-CyD (1/2) NF indicating that Metronidazole is totally in amorphous state. This XRD data also correlates with the visual observation of Metronidazole/HP-β-CyD (1/2) 15

solution at which the solution was clear and homogeneous without any indication of uncomplexed/undissolved Metronidazole crystals (Fig. 2c). The XRD is a useful technique provides useful information about inclusion complexation state of guest molecules with CyDs (Kayaci and Uyar, 2012, 2011; Narayanan et al., 2017). For instance, when the guest molecules are complexed within CyD cavity, they no longer form crystals as guest molecules are separated from each other by the CyD molecules (Kayaci and Uyar, 2012, 2011; Narayanan et al., 2017). Hence, the XRD data revealed that Metronidazole was totally amorphous by inclusion complexation with HP-β-CyD in Metronidazole/HP-β-CyD (1/2) NF sample. In the case of Metronidazole/HP-β-CyD (1/1) NF sample, most of the Metronidazole was in the amorphous state, yet, there was few amounts of uncomplexed Metronidazole crystals present in this sample. It is worth to mention that amorphization of drug molecules is desired for their fast-dissolution (Nagy et al., 2015; Seif et al., 2015; Yu et al., 2018). Here, the amorphous nature of Metronidazole encapsulated in HP-β-CyD fiber matrices in the form of inclusion complex along with highly porous and very large surface area of Metronidazole/HP-β-CyD NF webs greatly help the fast-dissolving behavior of these samples as discussed in the below section. The DSC thermograms of Metronidazole powder, pristine HP-β-CyD NF and Metronidazole/HP-β-CyD (1/1 and 1/2) NF samples are provided in Fig. 5b. DSC also provides very useful information about inclusion complexation state between guest molecules and host CyD molecules (Narayanan et al., 2017). For instance, since the guest molecules are in amorphous state in the case of inclusion complexation with CyD molecules, no melting point would be recorded for guest molecules from DSC thermogram of host-guest inclusion complexes (Celebioglu et al., 2016). The DSC thermogram of Metronidazole powder has a melting peak at 160 °C confirming that Metronidazole powder is crystalline Fig. 5b. The DSC thermogram of 16

pristine HP-β-CyD NF has a broad endothermic peak between 30-140 °C due to water loss (Celebioglu et al., 2016). The DSC thermograms of both Metronidazole/HP-β-CyD (1/1 and 1/2) NF samples were very similar to that of pristine HP-β-CyD NF sample showing only broad endothermic peak between 30-140 °C due to water loss, and did not have any melting peak for Metronidazole indicating that Metronidazole is in the amorphous state due to inclusion complexation. In XRD of Metronidazole/HP-β-CyD (1/1) NF, there were very low intensity diffraction peaks for the crystalline Metronidazole, but, no melting was observed in DSC of Metronidazole/HP-β-CyD (1/1) NF suggesting that the amount of Metronidazole crystals is very less and therefore the melting point for such low amount of Metronidazole crystals couldn’t be detected by DSC. Overall, there is a good agreement between DSC and XRD data suggesting that Metronidazole was inclusion complexed with HP-β-CyD and it was in the amorphous state in Metronidazole/HP-β-CyD NF samples. The thermo-analytic analyses of pure Metronidazole, pristine HP-β-CyD NF and Metronidazole/HP-β-CyD (1/1 and 1/2) NF were carried out by using TGA (Fig. 6). The Metronidazole indicates one-step mass loss at 262 °C. While HP-β-CyD NF exhibit two main weight losses from 25 °C and 400 °C due to the water loss (up to 100°C) and the main degradation of HP-β-CyD (max. temp 358 °C), four steps mass losses were observed in case of Metronidazole/HP-β-CyD (1/1 and 1/2) NF. The mass losses at the TGA thermograms of Metronidazole/HP-β-CyD (1/1 and 1/2) NF correspond to the water loss, main degradation of Metronidazole (two steps) and main degradation of HP-β-CyD. As seen from the derivative curves, the main degradation of Metronidazole occurs in two steps for both Metronidazole/HP-βCyD (1/1) NF and Metronidazole/HP-β-CyD (1/2) NF. The first degradation step of Metronidazole arises at lower temperature of 196 °C for Metronidazole/HP-β-CyD (1/1 and 1/2) 17

NF compared to pure Metronidazole powder (262 °C). The lower degradation temperature of Metronidazole in Metronidazole/HP-β-CyD (1/1 and 1/2) NF demonstrates the amorphous distribution of Metronidazole molecules in these samples contrasting its crystalline powder form (Celebioglu et al., 2016). It is also obvious from the derivate curves that there is another degradation step of Metronidazole between 260°C to 305°C for both Metronidazole/HP-β-CyD (1/1 and 1/2) NF which is partially buried under the main degradation of HP-β-CyD (Fig. 6b). This additional degradation step of Metronidazole in Metronidazole/HP-β-CyD (1/1 and 1/2) NF is possibly originated from stronger interactions between Metronidazole and HP-β-CyD. This types of increase in thermal stability of guest molecules is very common for inclusion complex systems and such improved thermal stability for the guest molecules is considered as an evidence of inclusion complexation (Narayanan et al., 2017). The TGA findings suggest that Metronidazole and HP-β-CyD form inclusion complexes upon different strengths of interactions in Metronidazole/HP-β-CyD (1/1 and 1/2) NF. TGA technique enables to calculate the components ratios in the samples. However, the amount of Metronidazole in Metronidazole/HPβ-CyD (1/1 and 1/2) NF could not be exactly calculated from the TGA thermograms because of the overlapped degradation steps. On the other hand, it was already calculated from the 1H-NMR analysis that, Metronidazole/HP-β-CyD (1/1) NF and Metronidazole/HP-β-CyD (1/2) NF were produced by the initial content of ~ 10 % (w/w) and ~ 6 % (w/w) Metronidazole, respectively used for the preparation of 1/1 and 1/2 molar ratio of Metronidazole/HP-β-CyD. Nevertheless, the TGA results indicated the inclusion complex formation between Metronidazole and HP-βCyD in Metronidazole/HP-β-CyD (1/1 and 1/2) NF by means of altered degradation temperature of Metronidazole molecules.

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3.3. Solubility, dissolution and disintegration profile of Hydrocortisone/HP-β-CyD NF webs Phase solubility test was performed to examine the solubilizing effect of HP-β-CyD on the Metronidazole molecules. Here, Metronidazole/HP-β-CyD solutions having different HP-β-CyD concentrations were stirred for 24 h to reach the complexation equilibrium. Then, each system was filtered to eliminate the undissolved Metronidazole in the solution and examined by UV-Vis spectroscopy technique. The phase solubility graph (Fig. 7a) indicates the solubility trend of Metronidazole against increasing HP-β-CyD concentrations from 0 to 120 mM. The solubility of Metronidazole (~ 54mM) was enhanced ~2 times in the 120 mM concentrated solutions of HP-βCyD due to the inclusion complex formation. As it was reported by Higuchi and Connors, there are different types of phase solubility diagrams depending on the guest molecules and CyD (Higuchi and Connors, 1965). A-type phase solubility profile is mostly observed in case of modified CyD systems and has subtypes of AP, AN and AL, and which stands for positively deviation of isotherms, negatively deviation of isotherms and linear increases in guest solubility as a function of CyD concentration, respectively (Higuchi and Connors, 1965). In our system, the phase solubility diagram indicates almost linear profile till the HP-β-CyD concentration of 100 mM. However, the highest HP-β-CyD concentration of 120 mM can be considered as the approximate limits for the solubilization of Metronidazole and phase solubility profile tends to adopt AN-type pattern by the highest HP-β-CyD concentration. The phase solubility diagram also enabled to calculate the stability constants of inclusion complex formed between Metronidazole and HP-β-CyD. Here, the apparent stability constant (Ks) was calculated using the straight-line portion of phase solubility diagram. Essentially Ks value represents the binding strength between guest molecules and CyD cavity. In our case, the Ks was calculated as 30 M-1 for

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Metronidazole/HP-β-CyD system and it is correlated with the Ks value reported in another study regarding the modified CyD/Metronidazole inclusion complexes (Malli et al., 2018). The comparison of in vitro dissolution profile of Metronidazole from Metronidazole/HPβ-CyD (1/1 and 1/2) NF webs and Metronidazole powder is shown in Fig. 7b. The Metronidazole/HP-β-CyD (1/1 and 1/2) NF webs disappeared instantly after they were contacted with the dissolution medium. Metronidazole/HP-β-CyD (1/1) NF and Metronidazole/HP-β-CyD (1/2) NF webs released 46.2±4.7 µg/mL and 38.5±1.5 µg/mL of Metronidazole in the first 30 seconds, respectively and both samples showed plateau profile up to 10 minutes, because during the rapid dissolution process of samples, the Metronidazole in nanofibrous webs simultaneously dissolved out and was free in the dissolution medium. On the other hand, Metronidazole powder did not dissolve immediately and only 9.1±1.7 µg/mL of Metronidazole was dissloved from the drug crystals into the dissolution medium in the first 30 seconds and it reached up to concentration of 33.3±2.6 µg/mL by the end of 10 minutes. This finding verified the improvement of the dissolution of Metronidazole in the Metronidazole/HP-β-CyD (1/1 and 1/2) NF webs compared to pure Metronidazole powder. The enhanced rapid dissolution profile of Metronidazole in Metronidazole/HP-β-CyD (1/1 and 1/2) NF webs can be ascribed to the essential properties of (i) inclusion complex formation between Metronidazole and HP-β-CyD; (ii) high water solubility of modified CyD types of HP-β-CyD; (iii) high surface area and high porosity of nanofibrous webs which provide an effective penetration path and higher amount of contact sides for the dissolution media. As seen in Fig. 7b, Metronidazole/HP-β-CyD (1/1) NF showed higher dissolution concentration compared to Metronidazole/HP-β-CyD (1/2) NF for the same sample amount (~ 40 mg) and this reflects the different Metronidazole content in inclusion complex nanofibers. For Metronidazole/HP-β-CyD (1/2) NF web, the dissolved Metronidazole

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concentration of 38.5±1.5 µg/mL is quite close to the theoretical and initial concentration of ~ 40 µg/mL for this sample. So, the fully amorphous state of Metronidazole in Metronidazole/HP-βCyD (1/2) NF web provided complete dissolution of drug in the aqueous medium. On the other hand, Metronidazole/HP-β-CyD (1/1) NF web showed lower dissolution profile (46.2±4.7 µg/mL) compared to its theoretical concentration (~ 80 µg/mL). This is possibly because of the presence of some uncomplexed of Metronidazole in Metronidazole/HP-β-CyD (1/1) NF which could not be dissolved in the aqueous medium in the given period of time. Nevertheless, both the inclusion complexation and the nanofibrous structure of Metronidazole/HP-β-CyD (1/1 and 1/2) NF webs facilitated the improvement of dissolution rate of Metronidazole, and our results suggested that Metronidazole/HP-β-CyD NF webs can be promising candidate for the fastdissolving oral drug delivery system. The water solubility of Metronidazole in Metronidazole/HP-β-CyD NF samples was also evaluated by the constant stirring for 24 hours at room temperature. The pure Metronidazole (~100 mM) and Metronidazole/HP-β-CyD (1/1 and 1/2) NF samples with the web concentration of ~ 0.3 g/mL were dissolved in water and then, each aqueous solution was filtered to remove the undissolved part of Metronidazole if any and then UV–Vis spectroscopy measurement was performed for these solutions. The recorded UV-Vis spectra of the aqueous solutions of Metronidazole and Metronidazole/HP-β-CyD (1/1 and 1/2) NF samples were given in Fig. 7c. Even though, the solution of Metronidazole and Metronidazole/HP-β-CyD (1/2) NF were prepared to have the same amount of Metronidazole (~100 mM), the absorption intensity of the solution of Metronidazole/HP-β-CyD (1/2) NF is higher than the pure Metronidazole solution due to the solubility increase of Metronidazole by HP-β-CyD inclusion complexation. On the other hand, Metronidazole/HP-β-CyD (1/1) NF solution had only slightly higher absorption

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intensity than Metronidazole/HP-β-CyD (1/2) NF. Actually, it was expected to observe more distinct difference of absorption intensity for the same web concentration (~ 0.3 g/mL) of Metronidazole/HP-β-CyD (1/1) NF compared to Metronidazole/HP-β-CyD (1/2) NF, since Metronidazole/HP-β-CyD (1/1) NF (~ 10 % (w/w)) sample has much higher Metronidazole content compared to Metronidazole/HP-β-CyD (1/2) NF (~ 6 % (w/w). However, as confirmed by the photo of electrospinning solution and XRD data, there was some undissolved part of Metronidazole crystals in case of Metronidazole/HP-β-CyD (1/1) NF web. The uncomplexed/ undissolved part of Metronidazole was most probably removed by the filtering before the UVVis measurement and therefore the solution of Metronidazole/HP-β-CyD (1/1) NF did not show an absorption intensity as high as expected. These findings are also correlated with the fast dissolution profiles of samples which were plotted for 10 minutes (Fig. 7b) and Metronidazole/HP-β-CyD (1/1) NF indicated lower dissolution concentration than the theoretical amount of Metronidazole present in Metronidazole/HP-β-CyD (1/1) NF web. Nevertheless, the result evidently showed that the inclusion complexation of Metronidazole with HP-β-CyD enhanced the water-solubility of Metronidazole in Metronidazole/HP-β-CyD (1/1 and 1/2) NF samples. The fast-dissolution of Metronidazole/HP-β-CyD (1/1 and 1/2) NF webs was visually tested by adding 3 mL of water to glass vials (Fig. 8a, Video S1). In order to have the same quantity of Metronidazole (~2 mg) in Metronidazole/HP-β-CyD NF webs, we placed ~19 mg of Metronidazole/HP-β-CyD (1/1) NF web and ~35 mg of Metronidazole/HP-β-CyD (1/2) NF web into separate vials. The same quantity of pure Metronidazole powder (~2 mg) was also placed into a glass vial and tested for comparison. Metronidazole is a poorly water-soluble drug; hence, the Metronidazole powder did not dissolve with the addition of water into the vial. The pristine 22

HP-β-CyD NF web (~35 mg) without Metronidazole was also tested as a control sample. The HP-β-CyD is a highly water soluble CyD type, therefore, the pristine HP-β-CyD NF web was dissolved immediately upon contact with water. Both Metronidazole/HP-β-CyD (1/1) NF and Metronidazole/HP-β-CyD (1/2) NF webs also disappeared immediately when water added to the vials. There was no indication of undissolved Metronidazole when both Metronidazole/HP-βCyD NF webs were dissolved confirming that the water solubility of Metronidazole was improved compared to its powder form. From XRD data, there was a few amounts of crystalline uncomplexed Metronidazole in Metronidazole/HP-β-CyD (1/1) NF web, and dissolution tests indicated that the uncomplexed part of Metronidazole in Metronidazole/HP-β-CyD (1/1) NF web could not dissolve in the dissolution medium. Even so, we didn’t detect any difference between Metronidazole/HP-β-CyD (1/1) NF web and Metronidazole/HP-β-CyD (1/2) NF web during the visual evaluation of fast-dissolution behavior. This might be due to the homogenous distribution of Metronidazole through the nanofibrous webs even though it is in uncomplexed/crystal form. The fast-disintegration of Metronidazole/HP-β-CyD (1/1 and 1/2) NF webs was also tested by contacting these webs to artificial saliva by using wet filter paper to mimic the moist environment of oral cavity (Bi et al., 1996). As recorded by video (Fig. 8b, Video S2), Metronidazole/HP-β-CyD (1/1 and 1/2) NF webs were very rapidly adsorbed by wet filter paper and dissolved instantly. HP-β-CyD is a highly water-soluble CyD derivative (Loftsson and Brewster, 2010), hence, HP-β-CyD as a fiber matrix would have a significant effect for the fastdisintegration of these Metronidazole/HP-β-CyD NF webs. More importantly, Metronidazole become readily water-soluble since it is inclusion complexed with HP-β-CyD in these Metronidazole/HP-β-CyD NF webs. Moreover, Metronidazole/HP-β-CyD NF webs have highly

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porous structure and very high surface area in which the liquid easily penetrates through and contact with the nanofibers and facilitate their fast-dissolution. 4. Conclusion Cyclodextrins are used as functional excipients in drug formulations since cyclodextrins can significantly enhance water solubility of poorly water-soluble drugs by inclusion complex formation. In this study, we produce Metronidazole/Hydroxypropyl-β-Cyclodextrin (HP-β-CyD) inclusion complex nanofibrous webs via electrospinning technique in order to have fastdissolving delivery of Metronidazole. We have chosen highly water soluble cyclodextrin type (Hydroxypropyl-beta-Cyclodextrin, HP-β-CyD) as a fiber matrix for encapsulation of Metronidazole, and more importantly HP-β-CyD also formed inclusion complex with poorly water-soluble Metronidazole in order to enhance its water solubility. For this purpose, the polymer-free electrospinning of Metronidazole/HP-β-CyD nanofibers (NF) was performed by using aqueous solutions of Metronidazole/HP-β-CyD inclusion complexes. The loading of Metronidazole can be adjusted in Metronidazole/HP-β-CyD NF since different molar ratio of Metronidazole/HP-β-CyD (e.g: 1/1 and 1/2) can be electrospun into bead-less fiber morphology. In addition, there was no loss of Metronidazole during the electrospinning process suggesting that the efficient encapsulation of Metronidazole within HP-β-CyD fiber matrix was achieved. The Metronidazole/HP-β-CyD (1/1 and 1/2) NF were in the form of nanofibrous webs with freestanding and flexible character and they were instantly dissolved when placed in water or contacted to artificial saliva. To conclude, the nanoporous structure of Metronidazole/HP-β-CyD NF webs can facilitate easy penetration of saliva in the oral cavity and high water-solubility of HP-β-CyD and inclusion complex state of Metronidazole with HP-β-CyD can offer fast-

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dissolution and immediate release of Metronidazole and therefore Metronidazole/HP-β-CyD NF webs can be quite appropriate as fast-dissolving oral drug delivery system.

Declaration of Competing Interest The Authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work made use of the Cornell Center for Materials Research Shared Facilities which are supported through the NSF MRSEC program (DMR-1719875), and the Cornell Chemistry NMR Facility supported in part by the NSF MRI program (CHE-1531632), and the Department of Fiber Science & Apparel Design facilities. Prof. Uyar acknowledges the startup funding from the College of Human Ecology at Cornell University. The partial funding for this research was also graciously provided by Nixon Family (Lea and John Nixon) thru College of Human Ecology at Cornell University. References Andersen, F.M., Bundgaard, H., 1984. Inclusion complexation of metronidazole benzoate with β-cyclodextrin and its depression of anhydrate-hydrate transition in aqueous suspensions. Int. J. Pharm. 19, 189–197. Aytac, Z., Ipek, S., Erol, I., Durgun, E., Uyar, T., 2019. Fast-dissolving electrospun gelatin nanofibers encapsulating ciprofloxacin/cyclodextrin inclusion complex. Colloids Surfaces B Biointerfaces 178, 129–136. Aytac, Z., Sen, H.S., Durgun, E., Uyar, T., 2015. Sulfisoxazole/cyclodextrin inclusion complex incorporated in electrospun hydroxypropyl cellulose nanofibers as drug delivery system. Colloids Surfaces B Biointerfaces 128, 331–338. Bala, R., Khanna, S., Pawar, P., Arora, S., 2013. Orally dissolving strips: A new approach to oral drug delivery system. Int. J. Pharm. Investig. https://doi.org/10.4103/2230-973x.114897

25

Balogh, A., Horváthová, T., Fülöp, Z., Loftsson, T., Harasztos, A.H., Marosi, G., Nagy, Z.K., 2015. Electroblowing and electrospinning of fibrous diclofenac sodium-cyclodextrin complex-based reconstitution injection. J. Drug Deliv. Sci. Technol. 26, 28–34. Bi, Y., Sunada, H., Yonezawa, Y., Danjo, K., Otsuka, A., IIDA, K., 1996. Preparation and evaluation of a compressed tablet rapidly disintegrating in the oral cavity. Chem. Pharm. Bull. 44, 2121–2127. Bilensoy, E., 2011. Cyclodextrins in pharmaceutics, cosmetics, and biomedicine: current and future industrial applications. John Wiley & Sons. Bottino, M.C., Arthur, R.A., Waeiss, R.A., Kamocki, K., Gregson, K.S., Gregory, R.L., 2014. Biodegradable nanofibrous drug delivery systems: effects of metronidazole and ciprofloxacin on periodontopathogens and commensal oral bacteria. Clin. Oral Investig. 18, 2151–2158. Bottino, M.C., Kamocki, K., Yassen, G.H., Platt, J.A., Vail, M.M., Ehrlich, Y., Spolnik, K.J., Gregory, R.L., 2013. Bioactive nanofibrous scaffolds for regenerative endodontics. J. Dent. Res. 92, 963–969. Brewster, M.E., Loftsson, T., 2007. Cyclodextrins as pharmaceutical solubilizers. Adv. Drug Deliv. Rev. 59, 645–666. Bukhary, H., Williams, G.R., Orlu, M., 2018. Electrospun fixed dose formulations of amlodipine besylate and valsartan. Int. J. Pharm. 549, 446–455. Canbolat, M.F., Celebioglu, A., Uyar, T., 2014. Drug delivery system based on cyclodextrinnaproxen inclusion complex incorporated in electrospun polycaprolactone nanofibers. Colloids Surfaces B Biointerfaces 115, 15–21. Carneiro, S.B., Duarte, C., Ílary, F., Heimfarth, L., Quintans, S., de Souza, J., Quintans-Júnior, L.J., Veiga Júnior, V.F. da, Neves de Lima, Á.A., 2019. Cyclodextrin–Drug Inclusion Complexes: In Vivo and In Vitro Approaches. Int. J. Mol. Sci. 20, 642. Celebioglu, A., Kayaci-Senirmak, F., İpek, S., Durgun, E., Uyar, T., 2016. Polymer-free nanofibers from vanillin/cyclodextrin inclusion complexes: high thermal stability, enhanced solubility and antioxidant property. Food Funct. 7, 3141–3153. Celebioglu, A., Uyar, T., 2019. Fast Dissolving Oral Drug Delivery System based on Electrospun Nanofibrous Webs of Cyclodextrin/Ibuprofen Inclusion Complex Nanofibers. Mol. Pharm. 16, 4387-4398. Celebioglu, A., Uyar, T., 2012. Electrospinning of nanofibers from non-polymeric systems: polymer-free nanofibers from cyclodextrin derivatives. Nanoscale 4, 621–631. Chowdary, Y.A., Soumya, M., Madhu Babu, M., Aparna, K., Himabindu, P., 2012. A review on fast dissolving drug delivery systems-a pioneering drug delivery technology. Bull Env Pharmacol Life Scien 1, 8–20. Flemmig, T.F., Milian, E., Kopp, C., Karch, H., Klaiber, B., 1998. Differential effects of 26

systemic metronidazole and amoxicillin on Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in intraoral habitats. J. Clin. Periodontol. 25, 1–10. Giram, P.S., Shitole, A., Nande, S.S., Sharma, N., Garnaik, B., 2018. Fast dissolving moxifloxacin hydrochloride antibiotic drug from electrospun Eudragit L-100 nonwoven nanofibrous Mats. Mater. Sci. Eng. C 92, 526–539. He, M., Jiang, H., Wang, R., Xie, Y., Zhao, C., 2017. Fabrication of metronidazole loaded poly (ε-caprolactone)/zein core/shell nanofiber membranes via coaxial electrospinning for guided tissue regeneration. J. Colloid Interface Sci. 490, 270–278. He, M., Xue, J., Geng, H., Gu, H., Chen, D., Shi, R., Zhang, L., 2015. Fibrous guided tissue regeneration membrane loaded with anti-inflammatory agent prepared by coaxial electrospinning for the purpose of controlled release. Appl. Surf. Sci. 335, 121–129. He, P., Zhong, Q., Ge, Y., Guo, Z., Tian, J., Zhou, Y., Ding, S., Li, H., Zhou, C., 2018. Dual drug loaded coaxial electrospun PLGA/PVP fiber for guided tissue regeneration under control of infection. Mater. Sci. Eng. C 90, 549–556. Higuchi, T., Connors, K.A., 1965. Phase solubility diagram. Adv Anal Chem Instrum 4, 117– 212. Illangakoon, U.E., Gill, H., Shearman, G.C., Parhizkar, M., Mahalingam, S., Chatterton, N.P., Williams, G.R., 2014. Fast dissolving paracetamol/caffeine nanofibers prepared by electrospinning. Int. J. Pharm. 477, 369–379. Jones, D.S., Muldoon, B.C.O., Woolfson, A.D., Andrews, G.P., Sanderson, F.D., 2008. Physicochemical characterization of bioactive polyacrylic acid organogels as potential antimicrobial implants for the buccal cavity. Biomacromolecules 9, 624–633. Kamocki, K., Nör, J.E., Bottino, M.C., 2015. Dental pulp stem cell responses to novel antibiotic‐containing scaffolds for regenerative endodontics. Int. Endod. J. 48, 1147–1156. Kathpalia, H., Gupte, A., 2013. An Introduction to Fast Dissolving Oral Thin Film Drug Delivery Systems: A Review. Curr. Drug Deliv. https://doi.org/10.2174/156720181006131125150249 Kayaci, F., Uyar, T., 2012. Encapsulation of vanillin/cyclodextrin inclusion complex in electrospun polyvinyl alcohol (PVA) nanowebs: Prolonged shelf-life and high temperature stability of vanillin. Food Chem. 133, 641–649. Kayaci, F., Uyar, T., 2011. Solid inclusion complexes of vanillin with cyclodextrins: their formation, characterization, and high-temperature stability. J. Agric. Food Chem. 59, 11772–11778. Kwak, H.W., Woo, H., Kim, I.-C., Lee, K.H., 2017. Fish gelatin nanofibers prevent drug crystallization and enable ultrafast delivery. RSC Adv. 7, 40411–40417. Li, X., Kanjwal, M.A., Lin, L., Chronakis, I.S., 2013. Electrospun polyvinyl-alcohol nanofibers as oral fast-dissolving delivery system of caffeine and riboflavin. Colloids Surfaces B 27

Biointerfaces 103, 182–188. Liang, A.C., Chen, L.-L.H., 2001. Fast-dissolving intraoral drug delivery systems. Expert Opin. Ther. Pat. 11, 981–986. Loftsson, T., Brewster, M.E., 2010. Pharmaceutical applications of cyclodextrins: basic science and product development. J. Pharm. Pharmacol. 62, 1607–1621. Malli, S., Bories, C., Ponchel, G., Loiseau, P.M., Bouchemal, K., 2018. Phase solubility studies and anti-Trichomonas vaginalis activity evaluations of metronidazole and methylated βcyclodextrin complexes: Comparison of CRYSMEB and RAMEB. Exp. Parasitol. 189, 72– 75. Manasco, J.L., Tang, C., Burns, N.A., Saquing, C.D., Khan, S.A., 2014. Rapidly dissolving poly (vinyl alcohol)/cyclodextrin electrospun nanofibrous membranes. RSC Adv. 4, 13274– 13279. Mano, F., Martins, M., Sá-Nogueira, I., Barreiros, S., Borges, J.P., Reis, R.L., Duarte, A.R.C., Paiva, A., 2017. Production of electrospun fast-dissolving drug delivery systems with therapeutic eutectic systems encapsulated in gelatin. AAPS PharmSciTech 18, 2579–2585. Monteiro, A.P.F., Rocha, C.M.S.L., Oliveira, M.F., Gontijo, S.M.L., Agudelo, R.R., Sinisterra, R.D., Cortés, M.E., 2017. Nanofibers containing tetracycline/β-cyclodextrin: Physicochemical characterization and antimicrobial evaluation. Carbohydr. Polym. 156, 417–426. Nagaraju, T., Gowthami, R., Rajashekar, M., Sandeep, S., Mallesham, M., Sathish, D., Shravan Kumar, Y., 2013. Comprehensive Review On Oral Disintegrating Films. Curr. Drug Deliv. https://doi.org/10.2174/1567201811310010016 Nagy, Z.K., Balogh, A., Démuth, B., Pataki, H., Vigh, T., Szabó, B., Molnár, K., Schmidt, B.T., Horák, P., Marosi, G., 2015. High speed electrospinning for scaled-up production of amorphous solid dispersion of itraconazole. Int. J. Pharm. 480, 137–142. Nam, S., Lee, J.-J., Lee, S.Y., Jeong, J.Y., Kang, W.-S., Cho, H.-J., 2017. Angelica gigas Nakai extract-loaded fast-dissolving nanofiber based on poly (vinyl alcohol) and Soluplus for oral cancer therapy. Int. J. Pharm. 526, 225–234. Narayanan, G., Boy, R., Gupta, B.S., Tonelli, A.E., 2017. Analytical techniques for characterizing cyclodextrins and their inclusion complexes with large and small molecular weight guest molecules. Polym. Test. 62, 402–439. Palasuk, J., Kamocki, K., Hippenmeyer, L., Platt, J.A., Spolnik, K.J., Gregory, R.L., Bottino, M.C., 2014. Bimix antimicrobial scaffolds for regenerative endodontics. J. Endod. https://doi.org/10.1016/j.joen.2014.07.017 Parkash, V., Maan, S., Deepika, Yadav, S., Hemlata, Jogpal, V., 2011. Fast disintegrating tablets: Opportunity in drug delivery system. J. Adv. Pharm. Technol. Res. https://doi.org/10.4103/2231-4040.90877 Passos, P.C., Moro, J., Barcelos, R.C.S., Da Rosa, H.Z., Vey, L.T., Bürguer, M.E., Maciel, R.M., 28

Danesi, C.C., Edwards, P.C., Bottino, M.C., 2019. Nanofibrous antibiotic‐eluting matrices: Biocompatibility studies in a rat model. J. Biomed. Mater. Res. Part B Appl. Biomater. Perioli, L., Ambrogi, V., Rubini, D., Giovagnoli, S., Ricci, M., Blasi, P., Rossi, C., 2004. Novel mucoadhesive buccal formulation containing metronidazole for the treatment of periodontal disease. J. Control. release 95, 521–533. Qin, Z., Jia, X.-W., Liu, Q., Kong, B., Wang, H., 2019. Fast dissolving oral films for drug delivery prepared from chitosan/pullulan electrospinning nanofibers. Int. J. Biol. Macromol. 137, 224–231. Quan, J., Yu, Y., Branford-White, C., Williams, G.R., Yu, D.-G., Nie, W., Zhu, L.-M., 2011. Preparation of ultrafine fast-dissolving feruloyl-oleyl-glycerol-loaded polyvinylpyrrolidone fiber mats via electrospinning. Colloids Surfaces B Biointerfaces 88, 304–309. Rahane, R.D., Rachh, P.R., 2018. A review on fast dissolving tablet. J. Drug Deliv. Ther. 8, 50– 55. Ramukutty, S., Ramachandran, E., 2012. Crystal growth by solvent evaporation and characterization of metronidazole. J. Cryst. Growth 351, 47–50. Reise, M., Wyrwa, R., Müller, U., Zylinski, M., Völpel, A., Schnabelrauch, M., Berg, A., Jandt, K.D., Watts, D.C., Sigusch, B.W., 2012. Release of metronidazole from electrospun poly (L-lactide-co-D/L-lactide) fibers for local periodontitis treatment. Dent. Mater. 28, 179– 188. Samprasit, W., Akkaramongkolporn, P., Kaomongkolgit, R., Opanasopit, P., 2018. Cyclodextrinbased oral dissolving films formulation of taste-masked meloxicam. Pharm. Dev. Technol. 23, 530–539. Schkarpetkin, D., Reise, M., Wyrwa, R., Völpel, A., Berg, A., Schweder, M., Schnabelrauch, M., Watts, D.C., Sigusch, B.W., 2016. Development of novel electrospun dual-drug fiber mats loaded with a combination of ampicillin and metronidazole. Dent. Mater. 32, 951–960. Seif, S., Franzen, L., Windbergs, M., 2015. Overcoming drug crystallization in electrospun fibers–Elucidating key parameters and developing strategies for drug delivery. Int. J. Pharm. 478, 390–397. Stjern, L., Voittonen, S., Weldemichel, R., Thuresson, S., Agnes, M., Benkovics, G., Fenyvesi, E., Malanga, M., Yannakopoulou, K., Feiler, A., 2017. Cyclodextrin-mesoporous silica particle composites for controlled antibiotic release. A proof of concept toward colon targeting. Int. J. Pharm. 531, 595–605. Topuz, F., Uyar, T., 2019. Electrospinning of Cyclodextrin Functional Nanofibers for Drug Delivery Applications. Pharmaceutics 11, 6. Uyar, T., Besenbacher, F., 2008. Electrospinning of uniform polystyrene fibers: The effect of solvent conductivity. Polymer (Guildf). 49, 5336–5343. Uyar, T., Kny, E., 2017. Electrospun materials for tissue engineering and biomedical 29

applications: research, design and commercialization. Woodhead Publishing. Vass, P., Démuth, B., Farkas, A., Hirsch, E., Szabó, E., Nagy, B., Andersen, S.K., Vigh, T., Verreck, G., Csontos, I., 2019. Continuous alternative to freeze drying: Manufacturing of cyclodextrin-based reconstitution powder from aqueous solution using scaled-up electrospinning. J. Control. Release 298, 120–127. Vigh, T., Horváthová, T., Balogh, A., Sóti, P.L., Drávavölgyi, G., Nagy, Z.K., Marosi, G., 2013. Polymer-free and polyvinylpirrolidone-based electrospun solid dosage forms for drug dissolution enhancement. Eur. J. Pharm. Sci. 49, 595–602. Wang, Yi, Jiang, Y., Zhang, Y., Wen, S., Wang, Yuguang, Zhang, H., 2019. Dual functional electrospun core-shell nanofibers for anti-infective guided bone regeneration membranes. Mater. Sci. Eng. C 98, 134–139. Xue, J., He, M., Liang, Y., Crawford, A., Coates, P., Chen, D., Shi, R., Zhang, L., 2014a. Fabrication and evaluation of electrospun PCL-gelatin micro-/nanofiber membranes for anti-infective GTR implants. J. Mater. Chem. B. https://doi.org/10.1039/c4tb00737a Xue, J., He, M., Liu, H., Niu, Y., Crawford, A., Coates, P.D., Chen, D., Shi, R., Zhang, L., 2014b. Drug loaded homogeneous electrospun PCL/gelatin hybrid nanofiber structures for anti-infective tissue regeneration membranes. Biomaterials. https://doi.org/10.1016/j.biomaterials.2014.07.060 Xue, J., He, M., Niu, Y., Liu, H., Crawford, A., Coates, P., Chen, D., Shi, R., Zhang, L., 2014c. Preparation and in vivo efficient anti-infection property of GTR/GBR implant made by metronidazole loaded electrospun polycaprolactone nanofiber membrane. Int. J. Pharm. 475, 566–577. Xue, J., Niu, Y., Gong, M., Shi, R., Chen, D., Zhang, L., Lvov, Y., 2015a. Electrospun microfiber membranes embedded with drug-loaded clay nanotubes for sustained antimicrobial protection. ACS Nano 9, 1600–1612. Xue, J., Shi, R., Niu, Y., Gong, M., Coates, P., Crawford, A., Chen, D., Tian, W., Zhang, L., 2015b. Fabrication of drug-loaded anti-infective guided tissue regeneration membrane with adjustable biodegradation property. Colloids Surfaces B Biointerfaces 135, 846–854. Yildiz, Z.I., Celebioglu, A., Uyar, T., 2017. Polymer-free electrospun nanofibers from sulfobutyl ether7-beta-cyclodextrin (SBE7-β-CD) inclusion complex with sulfisoxazole: Fastdissolving and enhanced water-solubility of sulfisoxazole. Int. J. Pharm. 531, 550–558. Yıldız, Z.I., Uyar, T., 2019. Fast-dissolving electrospun nanofibrous films of paracetamol/cyclodextrin-inclusion complexes. Appl. Surf. Sci. Yu, D.-G., Li, J.-J., Williams, G.R., Zhao, M., 2018. Electrospun amorphous solid dispersions of poorly water-soluble drugs: A review. J. Control. release. Yu, D.-G., Shen, X.-X., Branford-White, C., White, K., Zhu, L.-M., Bligh, S.W.A., 2009. Oral fast-dissolving drug delivery membranes prepared from electrospun polyvinylpyrrolidone ultrafine fibers. Nanotechnology 20, 55104. 30

Yuan, C., Liu, B., Liu, H., 2015. Characterization of hydroxypropyl-β-cyclodextrins with different substitution patterns via FTIR, GC–MS, and TG–DTA. Carbohydr. Polym. 118, 36–40. Zupančič, Š., Potrč, T., Baumgartner, S., Kocbek, P., Kristl, J., 2016. Formulation and evaluation of chitosan/polyethylene oxide nanofibers loaded with metronidazole for local infections. Eur. J. Pharm. Sci. 95, 152–160. Zupančič, Š., Preem, L., Kristl, J., Putrinš, M., Tenson, T., Kocbek, P., Kogermann, K., 2018. Impact of PCL nanofiber mat structural properties on hydrophilic drug release and antibacterial activity on periodontal pathogens. Eur. J. Pharm. Sci. 122, 347–358. Figure Captions Fig. 1. (a) The chemical structure of Metronidazole and HP-β-CyD. (b) The schematic representation of inclusion complex formation between Metronidazole and HP-β-CyD molecules, and (c) the electrospinning of Metronidazole/HP-β-CyD NF.

Fig. 2. The representative SEM images and the fiber diameter distribution graphs of (a-i) HP-βCyD NF, (b-i) Metronidazole/HP-β-CyD (1/1) NF and (c-i) Metronidazole/HP-β-CyD (1/2) NF. The photographs of the electrospinning solutions and the resulting electrospun nanofibrous webs of (a-ii) HP-β-CyD, (b-ii) Metronidazole/HP-β-CyD (1/1) and (c-ii) Metronidazole/HP-β-CyD (1/2).

Fig. 3. 1H-NMR spectra of (a) pure Metronidazole, (b) HP-β-CyD NF, (c) Metronidazole/HP-βCyD (1/1) NF and (d) Metronidazole/HP-β-CyD (1/2) NF. The 1H-NMR spectra were recorded by dissolving the samples in d6-DMSO. The characteristic peaks of Metronidazole and HP-βCyD are highlighted with blue and yellow color, respectively.

Fig. 4. The (a) The full and (b, c) expanded range FT-IR spectra of Metronidazole powder, HPβ-CyD NF, Metronidazole/HP-β-CyD (1/1) NF and Metronidazole/HP-β-CyD (1/2) NF. 31

Fig. 5. The (a) XRD pattern and (b) DSC thermograms of Metronidazole powder, HP-β-CyD NF, Metronidazole/HP-β-CyD (1/1) NF and Metronidazole/HP-β-CyD (1/2) NF.

Fig. 6. (a) TGA thermograms and (b) derivates of Metronidazole powder, HP-β-CyD NF, Metronidazole/HP-β-CyD (1/1) NF and Metronidazole/HP-β-CyD (1/2) NF. Fig. 7. (a) Phase solubility diagram of Metronidazole/HP-β-CyD inclusion complex system. (b) Time dependent dissolution profiles and (c) UV-Vis spectra of aqueous solutions of Metronidazole powder, Metronidazole/HP-β-CyD (1/1) and Metronidazole/HP-β-CyD (1/2) NF webs.

Fig.

8.

(a)

The

dissolution

behavior

of

Metronidazole

powder,

HP-β-CyD

NF,

Metronidazole/HP-β-CyD (1/1) NF and Metronidazole/HP-β-CyD (1/2) NF webs in distilled water. (b) The disintegration behavior of Metronidazole/HP-β-CyD (1/1) NF and Metronidazole/HP-β-CyD (1/2) NF webs at the artificial saliva environment. The pictures were captured from the videos which were given as Video S1 and Video S2.

32

33

Table 1. The solution properties and the fiber diameters of resulting electrospun nanofibers. Molar ration of

Viscosity

Conductivity

Average fiber

Metronidazole/HP-β-CyD

(Pa•s)

(µS/cm)

diameter (nm)

HPβCyD

-

1.533

36.3

220±60

Metronidazole/HP-β-CyD (1/1)

1/1

1.136

32.4

190±100

Metronidazole/HP-β-CyD (1/2)

1/2

1.121

33.9

195±80

Sample

34

35