Physico-Chemical, In Vitro and Ex Vivo Characterization of Meloxicam Potassium-Cyclodextrin Nanospheres

Nasal drug delivery has many beneficial properties, such as avoiding the first pass metabolism and rapid onset of action. However, the limited residence time on the mucosa and limited absorption of certain molecules make the use of various excipients necessary to achieve high bioavailability. The application of mucoadhesive polymers can increase the contact time with the nasal mucosa, and permeation enhancers can enhance the absorption of the drug. We aimed to produce nanoparticles containing meloxicam potassium (MEL-P) by spray drying intended for nasal application. Various cyclodextrins (hydroxypropyl-β-cyclodextrin, α-cyclodextrin) and biocompatible polymers (hyaluronic acid, poly(vinylalcohol)) were used as excipients to increase the permeation of the drug and to prepare mucoadhesive products. Physico-chemical, in vitro and ex vivo biopharmaceutical characterization of the formulations were performed. As a result of spray drying, mucoadhesive nanospheres (average particle size <1 µm) were prepared which contained amorphous MEL-P. Cyclodextrin-MEL-P complexes were formed and the applied excipients increased the in vitro and ex vivo permeability of MEL-P. The highest amount of MEL-P permeated from the α-cyclodextrin-based poly(vinylalcohol)-containing samples in vitro (209 μg/cm2) and ex vivo (1.47 μg/mm2) as well. After further optimization, the resulting formulations may be promising for eliciting a rapid analgesic effect through the nasal route.


Introduction
Nasal administration of different active pharmaceutical ingredients (APIs) is an investigated area, since it is possible not to just treat local pathological conditions, but to reach the systemic circulation or the central nervous system through the nasal epithelia because of its high vascularization and large surface area [1]. The absorbed drugs avoid the first pass hepatic metabolism and a rapid onset of action may occur [2]. Despite the many favorable properties, there are some limitations that need to be overcome for the success of drug absorption. Due to the mucociliary clearance, the nasal fluid renews every 15-20 min which results in short residence time of the API, and the low permeability of the mucosa is also a hindering factor [3,4]. By selecting the appropriate excipients-that are mucoadhesive (e.g., chitosan, hyaluronic acid, Carbopols [5]), so the contact time of the API is extended, or have permeability enhancing features (e.g., cyclodextrins, chitosan, clodextrin (HPβCD) and α-cyclodextrin (αCD) were from Cyclolab Ltd. ( gary). Hyaluronic acid (sodium salt) (HA) was from Contipro Biotech Czech Republic), poly(vinylalcohol) (PVA) and mucin (from porcine s were from Sigma-Aldrich (Sigma-Aldrich Co. LLC, St. Louis MO, US).

Preparation of the Spray Dried Samples
The solutions for nano spray drying were prepared by dissolving 1 of MEL-P and cyclodextrin (HPβCD or αCD) using 10 mL of distilled wat for the polymer-free samples. For the PVA-containing samples, 10 mg of to the solutions in addition to the aforementioned compounds, and for th samples, 5 mg of HA was dissolved in the MEL-P-cyclodextrin solutions ( Nano Spray Dryer B-90 HP (BÜCHI Labortechnik AG, Flawil, Switzerland used for the production of the samples. The following parameters were process: inlet air temperature: 80 °C, pump: 20%, aspirator capacity: 100% flow: 130 L·h −1 . The size, shape and surface morphology of the spray dried particles The size, shape and surface morphology of the spray dried particles were visualized by SEM (Hitachi S4700, Hitachi Scientific Ltd., Tokyo, Japan). Under an argon atmosphere, after sputter-coating the samples with gold-palladium in a high-vacuum evaporator, they were examined at 10 kV and 10 µA. The air pressure was 1.3-13 MPa. The size of the particles was measured by ImageJ program. From each sample, 100 particles were measured.

Differential Scanning Calorimetry (DSC)
Mettler Toledo DSC 821 e system and STAR e program V9.1 (Mettler Toledo Inc., Schwerzenbach, Switzerland) were used to implement the thermal analysis. Approximately 2-5 mg of samples in sealed aluminum pans were heated from 25 • C to 300 • C applying 10 • C·min −1 heating rate under a constant argon flow of 10 L·h −1 . Physical mixtures (PMs) of MEL-P, cyclodextrins, HA and PVA in the same ratio as the spray dried samples contained were mixed in a Turbula mixer (Turbula WAB, Systems Schatz, Muttenz, Switzerland) at 50 rpm for 10 min and were applied as control samples.

X-ray Powder Diffraction (XRPD)
To examine the physical state of MEL-P in the samples, XRPD was performed with a Bruker D8 Advance diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) with Cu K λI radiation (λ = 1.5406 Å). The samples were scanned at 40 kV and 40 mA with an angular range of 3-40 • 2θ. Si was used to calibrate the instrument. As controls, the PMs of MEL-P, cyclodextrins, HA and PVA were applied in the same ratio as the spray-dried samples contained were mixed in a Turbula mixer (Turbula WAB, Systems Schatz, Switzerland) at 50 rpm for 10 min. DIFFRACTPLUS EVA software was used to perform the manipulations: Kα2-stripping, background removal and smoothing.

Fourier-Transformed Infrared Spectroscopy (FT-IR)
The interactions between MEL-P and the excipients were investigated by the AVATAR330 FT-IR spectrometer (Thermo Nicolet, Unicam Hungary Ltd., Budapest, Hungary) in the interval of 400-4000 cm −1 , at an optical resolution of 4 cm −1 . Samples were grounded and compressed into pastilles at 10 t with 0.15 g of KBr.

Mucoadhesivity
The potential mucoadhesivity of the samples was estimated by the displacement of powders on the tilted surface of agar-mucin and-as controls-pure agar gels, using a protocol proposed in the literature [30]. Briefly, a hot solution of 2% agar with or without 2% mucin in phosphate buffer pH 6.4 was poured into a petri dish and left for gelation overnight. The gels were stored at 32 • C before the test. The 7.5 mg MEL-P-containing samples were placed on top of the gels in a spot with a diameter of approximately 10 mm. At the beginning of the investigation, the petri dishes were leaned at an angle of 45 • and the displacement of powder samples was measured against time. All measurements were conducted in triplicate.

In Vitro and Ex Vivo Permeability Studies
A modified horizontal diffusion model ( Figure 2) was applied to study the in vitro and ex vivo permeability of MEL-P [31]. This apparatus simulated the nasal conditions. The 7.5 mg of MEL-P-containing samples were added to the donor phase, which was 9 mL of SNES of pH 5.6 (represented the nasal fluid). Nine microliters of pH = 7.4 phosphate buffer-corresponding to the pH of the blood-was used as the acceptor phase. The temperature of the phases was 32 • C (Thermo Haake C10-P5, Sigma Aldrich Co.) and the rotation rate of the stir-bars was set to 300 rpm.
For the in vitro tests, the two chambers of the apparatus were divided by an artificial membrane (Whatman TM regenerated cellulose membrane filter with 0.45 µm pores) that was soaked in isopropyl myristate for 30 min before the investigation. It modeled the Pharmaceutics 2021, 13, 1883 5 of 14 lipophilic mucosa between the phases. For the ex vivo measurements, the permeability test was performed on human nasal mucosa (mucoperiostium) in the case of the formulations with the highest in vitro permeability. The pieces of the nasal mucosa for primary study were collected during daily clinical routine nasal and sinus surgeries (septoplasty, FESS) under general or local anesthesia. The surgical field was infiltrated with 1% Lidocain-Tonogen local injection and the mucosa was lifted from its base with a raspatorium or Cottle elevator. Transport from the operating room was performed in physiological saline.
The amount of MEL-P diffused to the acceptor phase was determined spectrophotometrically at 364 nm in real time with an AvaLight DH-S-BAL spectrophotometer (AVANTES, Apeldoorn, The Netherlands). Each measurement was carried out in triplicate. The flux was determined at 15 min and the permeation enhancement ratios for the in vitro measurements were calculated based on the following equations (Equations (1) and (2)) [32]: where P app is the apparent permeability coefficient (cm/s), Q is the total amount permeated throughout the incubation time (µg), A is the diffusion area of the artificial membrane (cm 2 ), c is the initial concentration of the drug in the donor compartment (µg/cm 3 ), and t is the total time of the experiment (s) and where R is the permeation enhancement ratio and P app (control) is the apparent permeability coefficient (cm/s) of MEL-P.
Tonogen local injection and the mucosa was lifted from its bas Cottle elevator. Transport from the operating room was performe The amount of MEL-P diffused to the acceptor phase was d metrically at 364 nm in real time with an AvaLight DH-S-(AVANTES, Apeldoorn, The Netherlands). Each measurement w cate. The flux was determined at 15 min and the permeation en in vitro measurements were calculated based on the following and Equation (2)) [32]: where Papp is the apparent permeability coefficient (cm/s), Q is the throughout the incubation time (μg), A is the diffusion area of (cm 2 ), c is the initial concentration of the drug in the donor comp is the total time of the experiment (s) and where R is the permeation enhancement ratio and Papp (control) bility coefficient (cm/s) of MEL-P.   Mitochondrial activity as a measure of cell viability was performed by MTT assay in 96-well cell culture microplates using RPMI 2650 cells (human nasal septum epithelial squamous carcinoma cells, obtained from American Type Culture Collection, ATCC, Manassas, VA, USA). RPMI 2650 cells were seeded at a density of 4 × 10 4 cells/well. First, serial dilution was made of the following stock concentrations: 4.11 mg/mL HPβCD_MEL-P_PVA_spd or 2.99 mg/mL αCD_MEL-P_PVA_spd or 5 µg/mL Lipopolysaccharide (LPS; ThermoFisher Scientific, Waltham, MA, USA), then the cells were incubated at 37 • C for 24 h. Later, 20 µL of thiazolyl blue tetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) was added to each well. After an additional incubation at 37 • C for 4 h, sodium dodecyl sulfate (Sigma-Aldrich, St. Louis, MO, USA) solution (10% in 0.01 M HCI) was added and were incubated overnight. Cytotoxicity of the compounds was then determined by measuring the OD at 550 nm (ref. 630 nm) with EZ READ 400 ELISA reader (Biochrom, Cambridge, UK). The assay was repeated four times for each concentration.

Examination of the Anti-Inflammatory Effect of Compounds in In Vitro Experiments
RPMI 2650 cells were seeded in 6-well plate at a density of 1 × 10 6 cells/well and treated with the highest noncytotoxic concentration either with 1.03 mg/mL HPβCD_MEL-P_PVA_spd and 5 µg/mL LPS or 0.38 mg/mL αCD_MEL-P_PVA_spd and 5 µg/mL LPS or 5 µg/mL LPS or left untreated. During the experiment the LPS was used as a positive control.

Total RNA Extraction and cDNA Synthesis
After 24 h treatment of RPMI 2650 cells with the compounds, RNA was extracted using the TRI reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's protocol. Subsequently, 1 µg of total RNA was reverse transcribed using Maxima Reverse Transcriptase (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions.

Particle Size and Morphology
SEM was used to visualize the morphology and particle size (PS) of the spray dried samples. The images revealed smooth surfaced, round shaped, spherical particles in all spray dried samples ( Figure 3) and the average PS measured by the ImageJ program was between 612 nm and 871 nm ( Table 2).

Thermal Properties
The DSC curves of the PMs and the spray dried samples are shown in Figure 4. The endothermic peaks at around 170 °C in the PMs are the melting points of raw MEL-P indicating its crystallinity [36]. The broad endothermic peaks from 40 °C to 105 °C in Figure 4a, and the endothermic bands from 60 °C to 105 °C shown in Figure 4b in the curves of the PMs are due to the dehydration of HPβCD and αCD, respectively [37][38][39]. In the case of the spray dried samples, besides the broad endothermic peaks caused by the loss of water, no thermal event could be observed. The disappearance of the endothermic peaks of MEL-P can suggest not only its amorphization, but the formation of inclusion complexes [40]. However, above 225 °C, the appearance of exotermic peaks in the PMs and in the products are due to the decomposition of MEL-P.

Thermal Properties
The DSC curves of the PMs and the spray dried samples are shown in Figure 4. The endothermic peaks at around 170 • C in the PMs are the melting points of raw MEL-P indicating its crystallinity [36]. The broad endothermic peaks from 40 • C to 105 • C in Figure 4a, and the endothermic bands from 60 • C to 105 • C shown in Figure 4b in the curves of the PMs are due to the dehydration of HPβCD and αCD, respectively [37][38][39]. In the case of the spray dried samples, besides the broad endothermic peaks caused by the loss of water, no thermal event could be observed. The disappearance of the endothermic peaks of MEL-P can suggest not only its amorphization, but the formation of inclusion complexes [40]. However, above 225 • C, the appearance of exotermic peaks in the PMs and in the products are due to the decomposition of MEL-P.

Thermal Properties
The DSC curves of the PMs and the spray dried samples are shown in Figure 4. The endothermic peaks at around 170 °C in the PMs are the melting points of raw MEL-P indicating its crystallinity [36]. The broad endothermic peaks from 40 °C to 105 °C in Figure 4a, and the endothermic bands from 60 °C to 105 °C shown in Figure 4b in the curves of the PMs are due to the dehydration of HPβCD and αCD, respectively [37][38][39]. In the case of the spray dried samples, besides the broad endothermic peaks caused by the loss of water, no thermal event could be observed. The disappearance of the endothermic peaks of MEL-P can suggest not only its amorphization, but the formation of inclusion complexes [40]. However, above 225 °C, the appearance of exotermic peaks in the PMs and in the products are due to the decomposition of MEL-P.

Structural Characterization
The XRPD diffractograms of the spray dried samples and the PMs are shown in Figure 5. The distinct peaks appearing at 6.1, 15.5, 24.6 and 30.9 2Θ values indicate the crystallinity of raw MEL-P in the PMs. In Figure 5a, the absence of characteristic peaks for HPβCD in the PMs suggesting its amorphous state. and in Figure 5b, the characteristic peaks at diffraction angles 2Θ of 5.3 • , 12.0 • , 14.4 • and 21.8 • indicate the crystallinity of αCD [41]. After spray drying, the intensity of the previously mentioned diffraction peaks assigned to MEL-P and αCD remarkably reduced referring to their amorphization and the formation of the inclusion complexes. These results corresponded to those of the thermal analysis. No change could be observed in the crystallinity of the products after three months of storage in laboratory conditions.

Structural Characterization
The XRPD diffractograms of the spray dried samples and the PMs are shown in Figure 5. The distinct peaks appearing at 6.1, 15.5, 24.6 and 30.9 2Θ values indicate the crystallinity of raw MEL-P in the PMs. In Figure 5a, the absence of characteristic peaks for HPβCD in the PMs suggesting its amorphous state. and in Figure 5b, the characteristic peaks at diffraction angles 2Θ of 5.3°, 12.0°, 14.4° and 21.8° indicate the crystallinity of αCD [41]. After spray drying, the intensity of the previously mentioned diffraction peaks assigned to MEL-P and αCD remarkably reduced referring to their amorphization and the formation of the inclusion complexes. These results corresponded to those of the thermal analysis. No change could be observed in the crystallinity of the products after three months of storage in laboratory conditions.

Secondary Interactions
The FT-IR spectra of raw HPβCD showed characteristic peaks at 1653 cm −1 (H-O-H bending), 2931 cm −1 (C-H stretching) and 3404 cm −1 (O-H stretching) [42]. In all of the HPβCD-based spray dried samples, the bands shifted to lower wavenumbers: 1653 cm −1 to 1616 cm −1 and 3404 cm −1 to 3385 cm −1 (Figure 6a). Considering raw αCD, the band at 3405 cm −1 shifted to lower wavenumbers (3385 cm −1 ) as well as in the αCD-based spray dried samples. In addition, wavenumber of band of αCD decreased from 1643 cm −1 to 1616 cm −1 , as well. These peaks are assigned to the stretching and the bending vibration of H-O in αCD, respectively (Figure 6b). The characteristic bands of HA and PVA were probably shaded in the spray dried samples by the other compounds. These changes can indicate the formation of hydrogen bonds between the MEL-P and the cyclodextrins.

Secondary Interactions
The FT-IR spectra of raw HPβCD showed characteristic peaks at 1653 cm −1 (H-O-H bending), 2931 cm −1 (C-H stretching) and 3404 cm −1 (O-H stretching) [42]. In all of the HPβCD-based spray dried samples, the bands shifted to lower wavenumbers: 1653 cm −1 to 1616 cm −1 and 3404 cm −1 to 3385 cm −1 (Figure 6a). Considering raw αCD, the band at 3405 cm −1 shifted to lower wavenumbers (3385 cm −1 ) as well as in the αCD-based spray dried samples. In addition, wavenumber of band of αCD decreased from 1643 cm −1 to 1616 cm −1 , as well. These peaks are assigned to the stretching and the bending vibration of H-O in αCD, respectively (Figure 6b). The characteristic bands of HA and PVA were probably shaded in the spray dried samples by the other compounds. These changes can indicate the formation of hydrogen bonds between the MEL-P and the cyclodextrins.

Structural Characterization
The XRPD diffractograms of the spray dried samples and the PMs are shown in Figure 5. The distinct peaks appearing at 6.1, 15.5, 24.6 and 30.9 2Θ values indicate the crystallinity of raw MEL-P in the PMs. In Figure 5a, the absence of characteristic peaks for HPβCD in the PMs suggesting its amorphous state. and in Figure 5b, the characteristic peaks at diffraction angles 2Θ of 5.3°, 12.0°, 14.4° and 21.8° indicate the crystallinity of αCD [41]. After spray drying, the intensity of the previously mentioned diffraction peaks assigned to MEL-P and αCD remarkably reduced referring to their amorphization and the formation of the inclusion complexes. These results corresponded to those of the thermal analysis. No change could be observed in the crystallinity of the products after three months of storage in laboratory conditions.

Secondary Interactions
The FT-IR spectra of raw HPβCD showed characteristic peaks at 1653 cm −1 (H-O-H bending), 2931 cm −1 (C-H stretching) and 3404 cm −1 (O-H stretching) [42]. In all of the HPβCD-based spray dried samples, the bands shifted to lower wavenumbers: 1653 cm −1 to 1616 cm −1 and 3404 cm −1 to 3385 cm −1 (Figure 6a). Considering raw αCD, the band at 3405 cm −1 shifted to lower wavenumbers (3385 cm −1 ) as well as in the αCD-based spray dried samples. In addition, wavenumber of band of αCD decreased from 1643 cm −1 to 1616 cm −1 , as well. These peaks are assigned to the stretching and the bending vibration of H-O in αCD, respectively (Figure 6b). The characteristic bands of HA and PVA were probably shaded in the spray dried samples by the other compounds. These changes can indicate the formation of hydrogen bonds between the MEL-P and the cyclodextrins.

Mucoadhesivity
The influence of HA and PVA on the potential mucoadhesivity of the samples was estimated. As controls, pure agar gels were applied, and all of the spray dried samples moved to the bottom of the petri dishes in the first minute of the investigation. In the case of the αCD-containing samples, the displacement of αCD_MEL-P_HA_spd was higher (3.3 cm) than the αCD_MEL-P_PVA_spd (2.69 cm), in 20 min. As an effect of the osmotic activity of HPβCD, HPβCD_MEL-P_HA_spd and HPβCD_MEL-P_PVA_spd samples adsorbed water from the gels resulting in their dissolution and complete displacement towards the bottom of the petri dishes in the first two minutes of the investigation. However, the PVA-containing sample moved slower. These results indicate higher mucoadhesivity of PVA compared to HA in the formulations.

In Vitro and Ex Vivo Permeability
For the in vitro test, the cumulative amount of MEL-P that diffused through the artificial membrane was measured as a function of time in a modified horizontal diffusion cell (Figure 7). The flux at 15 min ( Figure 8) and the enhancement ratios (Table 3) were determined. In the case of the HPβCD-based samples (Figure 7a), the highest amount of MEL-P permeated from the PVA-containing sample, where 194 µg/cm 2 of MEL-P diffused to the acceptor phase in 60 min. According to the enhancement ratio values, 7 times more drug permeated from this formulation, than of raw MEL-P. The same tendency could be observed in the case of the αCD-based samples (Figure 7b). The highest amount-209 µg/cm 2 in 60 min-of MEL-P permeated from the PVA-containing sample as well, more than 7.5 times more API could diffuse through the membrane. In both cases, the presence of PVA seemed to have a beneficial effect on the permeated amount of the drug. The same was experienced by Kaur, Indu P. et al., where the in vitro corneal permeation of acetazolamide was outstanding from their HPβCD-PVA-containing formulation [43]. According to literature data, this phenomenon presumably can be explained by the increasing effect of PVA on the free drug concentration in the aqueous diffusion layer on the surface of the biological and artificial membranes [43,44].
In contrast, the in vitro permeation enhancing effect of HA was not clearly convincing. Although the presence of HA was favorable in the HPβCD-based sample, in the case of the αCD-based sample, according to the enhancement ratio values, less amount of MEL-P diffused to the acceptor phase compared to the polymer-free formulation. In αCD_MEL-P_HA_spd, the deterioration of diffusion could occur due to the hindering effect of HA swelling retaining the release of MEL-P.
All of the prepared formulations provided higher in vitro permeation of the API than raw MEL-P; the use of PVA in the products seemed to have a distinctly beneficial effect on the amount of drug diffused under nasal conditions.

Mucoadhesivity
The influence of HA and PVA on the potential mucoadhesivity of the samples was estimated. As controls, pure agar gels were applied, and all of the spray dried samples moved to the bottom of the petri dishes in the first minute of the investigation. In the case of the αCD-containing samples, the displacement of αCD_MEL-P_HA_spd was higher (3.3 cm) than the αCD_MEL-P_PVA_spd (2.69 cm), in 20 min. As an effect of the osmotic activity of HPβCD, HPβCD_MEL-P_HA_spd and HPβCD_MEL-P_PVA_spd samples adsorbed water from the gels resulting in their dissolution and complete displacement towards the bottom of the petri dishes in the first two minutes of the investigation. However, the PVA-containing sample moved slower. These results indicate higher mucoadhesivity of PVA compared to HA in the formulations.

In Vitro and Ex Vivo Permeability
For the in vitro test, the cumulative amount of MEL-P that diffused through the artificial membrane was measured as a function of time in a modified horizontal diffusion cell (Figure 7). The flux at 15 min ( Figure 8) and the enhancement ratios (Table 3) were determined. In the case of the HPβCD-based samples (Figure 7a), the highest amount of MEL-P permeated from the PVA-containing sample, where 194 µg/cm 2 of MEL-P diffused to the acceptor phase in 60 min. According to the enhancement ratio values, 7 times more drug permeated from this formulation, than of raw MEL-P. The same tendency could be observed in the case of the αCD-based samples (Figure 7b). The highest amount-209 µg/cm 2 in 60 min-of MEL-P permeated from the PVA-containing sample as well, more than 7.5 times more API could diffuse through the membrane. In both cases, the presence of PVA seemed to have a beneficial effect on the permeated amount of the drug. The same was experienced by Kaur, Indu P. et al., where the in vitro corneal permeation of acetazolamide was outstanding from their HPβCD-PVA-containing formulation [43]. According to literature data, this phenomenon presumably can be explained by the increasing effect of PVA on the free drug concentration in the aqueous diffusion layer on the surface of the biological and artificial membranes [43,44].   In contrast, the in vitro permeation enhancing effect of HA was not clearly convincing. Although the presence of HA was favorable in the HPβCD-based sample, in the case of the αCD-based sample, according to the enhancement ratio values, less amount of MEL-P diffused to the acceptor phase compared to the polymer-free formulation. In αCD_MEL-P_HA_ spd, the deterioration of diffusion could occur due to the hindering effect of HA swelling retaining the release of MEL-P.
All of the prepared formulations provided higher in vitro permeation of the API than raw MEL-P; the use of PVA in the products seemed to have a distinctly beneficial effect on the amount of drug diffused under nasal conditions. The flux at 15 min followed a similar tendency as the permeation sequence ( Figure  8). The αCD_MEL-P_PVA_spd tended to diffuse the quickest through the membrane in vitro.
For the ex vivo measurements, the two PVA-containing samples, HPβCD_MEL-P_PVA_spd and αCD_MEL-P_PVA_spd were tested (Figure 9), because they showed the best in vitro results. The highest amount of MEL-P permeated from the αCD-based sample, where 1.47 µg/mm 2 MEL-P diffused into the acceptor phase in 60 min. From the HPβCD_MEL-P_PVA_spd sample, only less than a third of the aforementioned amount of MEL-P, 0.45 µg/mm 2 , permeated to the acceptor phase in 60 min. This phenomenon can be corresponding to the potential higher permeability enhancing effect of αCD than HPβCD by interacting with membrane phospholipids in the human nasal mucosal cells [45]. These results corresponded to those of the in vitro measurements.  The flux at 15 min followed a similar tendency as the permeation sequence ( Figure 8). The αCD_MEL-P_PVA_spd tended to diffuse the quickest through the membrane in vitro.
For the ex vivo measurements, the two PVA-containing samples, HPβCD_MEL-P_PVA_spd and αCD_MEL-P_PVA_spd were tested (Figure 9), because they showed the best in vitro results. The highest amount of MEL-P permeated from the αCD-based sample, where 1.47 µg/mm 2 MEL-P diffused into the acceptor phase in 60 min. From the HPβCD_MEL-P_PVA_spd sample, only less than a third of the aforementioned amount of MEL-P, 0.45 µg/mm 2 , permeated to the acceptor phase in 60 min. This phenomenon can be corresponding to the potential higher permeability enhancing effect of αCD than HPβCD by interacting with membrane phospholipids in the human nasal mucosal cells [45]. These results corresponded to those of the in vitro measurements.

In Vitro Cytotoxicity and IL-6, COX-2, IL-1b Expression
Cytotoxicity measurement revealed that the noncytotoxic concentrations are 1.03 mg/mL HPβCD_MEL-P_PVA_spd and 0.38 mg/mL αCD_MEL-P_PVA_spd. These results corresponded to the literature data, where a higher tolerable concentration was detected for HPβCD than αCD [46]. LPS was not cytotoxic to the cells.

In Vitro Cytotoxicity and IL-6, COX-2, IL-1b Expression
Cytotoxicity measurement revealed that the noncytotoxic concentrations are 1 mg/mL HPβCD_MEL-P_PVA_spd and 0.38 mg/mL αCD_MEL-P_PVA_spd. These sults corresponded to the literature data, where a higher tolerable concentration was tected for HPβCD than αCD [46]. LPS was not cytotoxic to the cells.

In Vitro Cytotoxicity and IL-6, COX-2, IL-1b Expression
Cytotoxicity measurement revealed that the noncytotoxic concentrations are 1 mg/mL HPβCD_MEL-P_PVA_spd and 0.38 mg/mL αCD_MEL-P_PVA_spd. These sults corresponded to the literature data, where a higher tolerable concentration was tected for HPβCD than αCD [46]. LPS was not cytotoxic to the cells.

Conclusions
In this work, MEL-P-containing particles were prepared by nano spray drying us HPβCD and αCD as permeability enhancers, and HA and PVA as mucoadhesive exc ents to obtain appropriate formulations for nasal administration. Using these additi MEL-P-cyclodextrin and MEL-P-cyclodextrin-polymer nanospheres were formulated study the effect of the type of cyclodextrin and the polymer in the composition on performance of the samples. The physico-chemical characterization, mucoadhesivity t in vitro permeability and cytotoxicity and ex vivo permeability studies were carried In all cases, nanospheres were successfully prepared (average PS <1 µm) while ME was present in mostly an amorphous state confirmed by DSC and XRPD. Secondary teractions were formed between the API and the cyclodextrins in each product, indica the complexation of MEL-P. The PVA-containing samples showed higher mucoadhesi than HA-containing ones, and among all the products, αCD_MEL-P_PVA_spd had potential highest mucoadhesive property according to our results. The in vitro flux a min was higher from HPβCD-containing samples than from αCD-containing ones, except for the PVA-containing products, the permeation extent from the αCD_M P_PVA_spd was the highest (209 µg/cm 2 in 60 min) among the samples. The presenc HA resulted in the decrease of in vitro permeation of MEL-P due to the retaining effec HA swelling, and meanwhile the presence of PVA caused remarkable increase of the p meation rate in the case of both cyclodextrins in vitro. The difference between αCD_MEL-P_PVA_spd and the HPβCD_MEL-P_PVA_spd samples was more noticea ex vivo (1.47 µg/mm 2 and 0.45 µg/mm 2 , respectively), which can be due to the higher p meation enhancing feature of αCD compared to HPβCD on nasal epithelial cells; howe HPβCD_MEL-P_PVA_spd had a higher tolerable concentration according to the cyto icity measurement. The two aforementioned formulations showed significant (p < 0.0 anti-inflammatory effect. Overall, αCD_MEL-P_PVA_spd showed the best results o the products based on our measurements.
The prepared formulations may be suitable for rapid onset of analgesic effect o adjuvants to opioids through the nasal route.

Conclusions
In this work, MEL-P-containing particles were prepared by nano spray drying using HPβCD and αCD as permeability enhancers, and HA and PVA as mucoadhesive excipients to obtain appropriate formulations for nasal administration. Using these additives, MEL-P-cyclodextrin and MEL-P-cyclodextrin-polymer nanospheres were formulated to study the effect of the type of cyclodextrin and the polymer in the composition on the performance of the samples. The physico-chemical characterization, mucoadhesivity test, in vitro permeability and cytotoxicity and ex vivo permeability studies were carried out. In all cases, nanospheres were successfully prepared (average PS <1 µm) while MEL-P was present in mostly an amorphous state confirmed by DSC and XRPD. Secondary interactions were formed between the API and the cyclodextrins in each product, indicating the complexation of MEL-P. The PVA-containing samples showed higher mucoadhesivity than HA-containing ones, and among all the products, αCD_MEL-P_PVA_spd had the potential highest mucoadhesive property according to our results. The in vitro flux at 15 min was higher from HPβCD-containing samples than from αCD-containing ones, and except for the PVA-containing products, the permeation extent from the αCD_MEL-P_PVA_spd was the highest (209 µg/cm 2 in 60 min) among the samples. The presence of HA resulted in the decrease of in vitro permeation of MEL-P due to the retaining effect of HA swelling, and meanwhile the presence of PVA caused remarkable increase of the permeation rate in the case of both cyclodextrins in vitro. The difference between the αCD_MEL-P_PVA_spd and the HPβCD_MEL-P_PVA_spd samples was more noticeable ex vivo (1.47 µg/mm 2 and 0.45 µg/mm 2 , respectively), which can be due to the higher permeation enhancing feature of αCD compared to HPβCD on nasal epithelial cells; however, HPβCD_MEL-P_PVA_spd had a higher tolerable concentration according to the cytotoxicity measurement. The two aforementioned formulations showed significant (p < 0.001) anti-inflammatory effect. Overall, αCD_MEL-P_PVA_spd showed the best results of all the products based on our measurements.
The prepared formulations may be suitable for rapid onset of analgesic effect or as adjuvants to opioids through the nasal route.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Medical Research Council (approval number: IV/3880-1/2021/EKU; date of approval: 4 May 2021).

Informed Consent Statement:
The ex vivo study was carried out on human nasal mucosal pieces. The mucosal pieces were collected via daily clinical routine surgeries from subjects who gave their informed consent for inclusion before they participated in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.