Based on previous studies, high molecular weight grades of Klucel^TM hydroxypropylcellulose (HPC) were used as an thermoplastic polymer to develope the bioadhesive hot melt extruded formulations for vaginal delivery [13] [14] . TEC, PEG400 were chosen as plasticizer. Firstly, HPC and PEG400/TEC were mixed using a pestle and a mortar and then natamycin were added to this mixture (Table 1). The extrusion temperature is preferred as 90˚C with a screw speed of 25 rpm. This raw material was fed into the hopper and transferred inside the heated barrel by rotating screws. The raw material was extruded with a co-rotating twin screw hot-melt extruder (Minilab HAAKE Rheomex CTW5, Thermo Fisher Scientific, Karlsruhe, Germany). The die diameter was preferred as 1.75 mm. The torque values were determined.

Melt viscosity values were measured by hot melt extruder automotically during the manufacturing process. The torque values were revealed depends on extrussion temperature and screw rotation speed. The following table consists of these values.

The thermal stabilities of natamycin was determined by the thermogravimetric analysis method (TGA) (Perkin Elmer TGA-4000). Sample weighed approximately 3 mg were placed in a porcelain bowl and heated at a rate of 10˚C/min between 30˚C - 400˚C. The change in weight was determined.

Extrudates of 5 mm length were used in drug release studies. The drug release was determined using 250 mL of pH 4.5 citrate buffer containing 0.1% SDS; 70 rpm; 37˚C in a horizontal shaker incubator (Thermo Fisher Scientific Max Q™ 6000) according to the sink conditions. At predetermined time intervals, samples were collected and assayed by using UPLC (Accela, Thermo Fisher Scientific, Karlsruhe, Germany) at 304 nm. The release medium was replaced with fresh medium every 24 hours.

The mucoadhesive strength of the formulations was evaluated by measuring the force required to detach the formulation from a 8% mucin solution using a 500 g load cell TPA in tension mode. The extrudates were attached to the lower end of the probe (P 10 Perspex, θ: 10 mm). The probe holding the extrudate was lowered on to the surface of the mucin with a constant speed of 0.1 mm・s⁻¹ and a contact force of 0.005 N were applied. After keeping in contact surfaces for 180 s, the probe was then moved vertically upward at a constant speed of 0.1 mm・s⁻¹. Maximum detachment force (F) was obtained from the force-distance graph. The area under the curve (AUC) was calculated from force-distance plot as the mucoadhesion (M). The tests were conducted at 37˚C and each experiment was carried out three times [15] .

The formulations and the processing conditions for the preparation of the vaginal bioadhesive mini matrices containing natamycin are listed in Table 1. HPC was chosen as the matrix-forming polymer since it is the only water-soluble cellulose derivative that is thermoplastic. HPC has a softening temperature in the range of 100˚C - 150˚C, depending on its molecular weight [16] . PEG400/TEC were used as plasticiser to facilitate the hot-melt extrusion process. Also, plasticisers provide die lubrication, reduce melt viscosity and improve melt uniformity [17] . Therefore, the extrudates could be produced at 90˚C successfully.

In this study, four formulations have been tried but a colour change has been detected at F4 and it was concluded that drug loadings up to 40% could be extruded. So the characterization studies have been revealed with F1, F2, F3.

Pharmaceutical polymers utilized in hot-melt extrusion typically require a plasticizer in order to reduce the Tg of the polymer and to facilitate the HME process [8] . According to the obtained results of melt viscosity measurements, the maximum torque values of F1 and F2 were close to each other and the mini matrices containing NT and HPC could be produced with low torque values using both of the plasticisers. However the maximum torque of extrudates prepared using PEG400 increased with increasing drug loading (increased resistance to the flow of dispersed drug particles) [7] (Table 2).

The results obtained from the TGA studies showed that natamisin rapidly degraded from 198˚C and weight loss increased (Figure 1).

Comparing the release profiles of F1 and F2, the release profile of the extrudate containing PEG 400 is closer to the zero order release (Figure 2). F2 has 2 hours

lag time, and it released approximately 5% and 23% of drug at the 8. and 24. hours respectively, whereas F1 reached 4% and 17% at the same time zones. Formulation F1 has just 1 hour lag time and its release is faster than formulation F2 for the first 4 hours. However, it was seen that the release rate of F2 is higher than formulation F1 up to 48 hour and than the release rates of two formulations were almost same.

Comparing the release profiles of F2 and F3, it was seen that drug release rate increased with increasing drug loading (Figure 3). The lag time is shortened on the extrudates containing 40% NT. First 8 hours, F3 has faster release than F2. At the 24 hour, F2 has slightly more release rate than F3 and later two of formulations showed same release rates. According to the in vitro drug release results, the release was extended up to 72 hours.

The mucoadhesion results work of mucoadhesion and peak detachment force were calculated from the resulting force-distance plot obtained from the mucoadhesion test and given in Figure 4.

The values of work of mucoadhesions were found as 771,977 mN・mm, 753,199 mN・mm, 686,356 mN・mm and peak detachment force values were found as 830,574 mN, 786,693 mN, 783,931 mN for the F1, F2, F3 respectively. These results showed that, the convenient mucoadhesion for the vaginal mucosa could be obtained by using hot melt extruded mini matrices produced with the matrix-forming bioadhesive polymer HPC. Additionally, it was observed that with increasing of natamycin ratio in the formulations, mucoadhesion was decreased.