*2.4. Optimization of In Situ Thermogelling Carrier System* 2.4.1. Rheological Studies

The rheological measurements were carried out with a Physica MCR302 rheometer (Anton Paar, Graz, Austria). A cone and plate type measuring device with cone angle of 1 ◦ was applied; the diameter of the cone was 25 mm, and the gap height in the middle of the cone was 0.046 mm. The gelation temperature was measured while the temperature was increased from 20 to 40 ◦C, using 1 ◦C/min heating rate. The measurement was performed at a constant frequency of 1.0 rad/min and at a constant strain of 1%. The gelation time of the polymer solutions was followed at a constant frequency of 1.0 rad/min and at a constant strain of 1% at 37 ◦C. The samples were stored at 5 ± 1 ◦C and taken immediately before the measurement. Viscoelastic character was determined by frequency sweep tests immediately after the gelation measurement, with a strain of 1% at 37 ◦C. Storage modulus (G'), loss modulus (G") and loss factor were determined over the angular frequency range from 0.1 to 100 rad/s. The applied strain value (1%) was in the range of the linear viscoelasticity of the gels.

#### 2.4.2. Muco-Adhesion Measurement

Muco-adhesion was analyzed by means of tensile tests (TA-XT Plus texture analyzer (Metron Kft, Budapest, Hungary)) equipped with a 5-kg load cell. As a simulated mucosal membrane, a filter paper (Whatman® qualitative filter paper, Sigma Aldrich Co. Ltd., Budapest, Hungary) with 25 mm diameter, impregnated with 50 µL of an 8% *w*/*w* mucin dispersion, was used, prepared with a simulated nasal electrolyte solution (SNES) consisting of 8.77 g/L sodium chloride (NaCl), 2.98 g/L potassium chloride (KCl), 0.59 g/L anhydrous calcium chloride (CaCl2) and dissolved in purified water; the pH was adjusted to 5.6 with 0.1 M HCl [12]. Five parallel measurements were performed. 20 mg of the sample was attached to the cylinder probe and placed in contact with the filter paper wetted with mucin. A 2500 mN preload was used for 3 min, then the cylinder probe was moved upwards to separate the sample from the substrate at a prefixed speed of 2.5 mm/min. The maximum detachment force (adhesive force) and the work of adhesion (A, mN/mm) were measured, the latter calculated as the area (AUC) under the "force versus distance" curve using the Exponent Connect software of the instrument. The formulations were thermostated at 37 ◦C for 30 min before measurement. As a reference system, 0.5% *w*/*w* NaHA aqueous solution was applied.

#### 2.4.3. Characterization of Nanoparticles

The formulations were characterized according to their average hydrodynamic diameter (Z-average), polydispersity index (PdI) and zeta potential using a Malvern Zeta sizer Nano ZS (Malvern Instruments, Worcestershire, UK) at 25 and 35 ◦C in folded capillary cells. The refractive index was set to 1.72. The pH of formulations was measured applying a WTW® inoLab® pH 7110 laboratory pH tester (Thermo Fisher Scientific, Budapest, Hungary). The osmolality of formulations was determined by osmometer (Knauer Semimicro Osmometer, Berlin, Germany) based on the freezing point depression method. Each measurement was carried out in triplicate and data are shown as means ± SD. The encapsulation efficiency and loading capacity of gel-embedded nanoparticles were prepared with a Hermle Z323K high performance refrigerated centrifuge (Hermle AG, Gossheim, Germany) at 17.500 rpm, 4 ◦C for 30 min. The amount of free MEL in the supernatant was determined by high performance liquid chromatography (HPLC). Encapsulation efficiency (EE) and loading capacity (LC) of formulations were calculated according to the following equations [24]:

$$EE\left(\%\right) = \frac{A\text{amount of drug applied} - A\text{amount of drug in the supernovaant}}{A\text{amount of drug applied}} \cdot 100\tag{1}$$

$$L\mathbb{C}\left(\%\right) = \frac{\text{Mass of drug encapulated}}{\text{Mass of nonparticles}} \cdot 100\tag{2}$$

The distribution of FITC labelled HSA-MEL nanoparticles in gel structure was visualized by a Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems GmbH, Wetzlar, Germany) and Visitron spinning disk confocal system (Visitron Systems GmbH, Puchheim, Germany). The P407 containing formulations and the FITC labelled HSA-MEL colloidal solution as reference were dropped onto slides and incubated for 10 min at 35 ◦C for thermo-gelling. Then, slides were excited with a 488 nm Argon laser, and fluorescence was detected with a 505 to 570 nm BP filter.

#### 2.4.4. Rapid Equilibrium Dialysis (RED)

In order to investigate the in vitro dissolution kinetics and release profile of different MEL-HSA-P407 formulations at nasal conditions, the RED Device (Thermo ScientificTM, Waltham, MA, USA) was used. A suspension of MEL was prepared in a phosphate buffer saline (PBS, pH 5.6) with a nominal concentration of 2 mg/mL as a control for the study. Both the control and in situ gelling MEL-HSA formulations were homogenized using an Eppendorf MixMate (Thermo ScientificTM, Waltham, MA, USA) vortex mixer for 30 s and an ultrasonic bath (Sonorex Digiplus, Bandelin GmbH & Co. KG, Berlin, Germany) for 10 min. The RED Device inserts (8K MWCO) were fitted into the PTFE base plate, then 150 µL of samples was placed into the donor chambers, while 300 µL of PBS (pH 5.6) was added to the acceptor chambers. Thereafter, the RED unit was covered with a sealing tape and incubated above gelling temperature (37 ◦C) on an orbital shaker (at 350 rpm) for 4 h. 50 µL aliquots were withdrawn from the acceptor chamber at 5, 15, 30, 60, 120 and 240 min time points and immediately replaced with the same amount of fresh medium. 50 µL of acetonitrile was added to the withdrawn samples and the MEL content was determined using HPLC. Five parallel measurements were performed.

#### 2.4.5. High Performance Liquid Chromatography (HPLC)

The determination of MEL concentration was performed with an Agilent 1260 HPLC (Agilent Technologies, Santa Clara, CA, USA). A Kinetex® C18 column (5 µm, 150 mm × 4.6 mm (Phenomenex, Torrance, CA, USA)) was used as stationary phase. The mobile phases consisted of 0.065 M KH2PO<sup>4</sup> aqueous solution adjusted to pH = 2.8 with phosphoric acid (A), and methanol (B). A linear gradient from 50–50% to 25–75% (A-B eluent) was applied from 0 to 14 min. Then, from 14 to 20 min the phase composition was set back to 50–50% A-B. Separation was performed at 30 ◦C with 1 mL/min flow rate. 10 µL of the samples was injected to determine the MEL's concentration at 355 ± 4 nm using the UV-VIS diode array detector. Data were evaluated using ChemStation B.04.03. Software (Agilent Technologies, Santa Clara, CA, USA). The retention time of MEL was observed at 14.34 min. The regression coefficient (R<sup>2</sup> ) of the calibration curve was 0.999 in the concentration range 1–200 µg/mL. The determined limits of detection (LOD) and quantification (LOQ) of MEL were 16 ppm and 49 ppm, respectively.

#### 2.4.6. In Vitro BBB Permeability Assay

Parallel artificial membrane permeability assay (PAMPA) was used to determine the brain specific effective permeability of MEL from the reference suspension and the MEL-HAS-P407 formulations [25]. The filter donor plate (Multiscreen™-IP, MAIPN4510, pore size 0.45 µm; Millipore, Merck Ltd., Budapest, Hungary) was coated with 5 µL of lipid solution containing 16 mg brain polar lipid extract (porcine) and 8 mg cholesterol dissolved

in 600 µL dodecane. The Acceptor Plate (MSSACCEPTOR; Millipore, Merck Ltd., Budapest, Hungary) was filled with 300 µL of a PBS solution of pH 7.4. 150–150 µL of the formulation and the reference solutions were applied on the membrane of the donor plate. Then, this was covered with a plate lid in order to decrease the possible evaporation of the solvent. This sandwich system was incubated at 37 ◦C for 4 h (Heidolph Titramax 1000, Heidolph Instruments, Schwabach, Germany). The concentration of MEL permeated in the acceptor plate was determined using HPLC. The effective permeability and membrane retention of drugs were calculated using the following equation [25]:

$$P\_{\varepsilon} \left( \text{cm/s} \right) = -\frac{2.303 \cdot V\_A}{A \left( t - \tau\_{SS} \right)} \cdot \log \left[ 1 - \frac{c\_A(t)}{S} \right] \tag{3}$$

where *P<sup>e</sup>* is the effective permeability coefficient (cm/s), *A* is the filter area (0.24 cm<sup>2</sup> ), *V<sup>A</sup>* is the volume of the acceptor phase (0.3 mL), *t* is the incubation time (s), *τSS* is the time to reach the steady state (s), *cA*(*t*) is the concentration of the compound in the acceptor phase at time point *t* (mol/mL), and *S* (mol/mL) is the solubility of MEL in the donor phase. The latter was determined after centrifugation (at 12000 rpm, 15 min, Eppendorf Centrifuge 5804 R) in Microcon Centrifugal Filter Devices (30,000 MWCO) and 50-times dilution of the formulations, using the same HPLC system. The flux of samples was calculated using the following equation [26]:

$$\text{Flux } (\text{mol/cm}^2 \cdot \text{s}) = P\_\text{\textdegree S} \tag{4}$$
