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Back to Journal »International Journal of Nanomedicine» Volume 15

Brain targeting of duloxetine hydrochloride by intranasal delivery of loaded cubic gel: in vitro characterization, in vitro permeation and in vivo biodistribution studies

Author Elsenosy FM, Abdelbary GA, Elshafeey AH, Elsayed I, Fares AR

The 2020 volume will be published on November 30, 2020: 15 pages 9517-9537

DOI https://doi.org/10.2147/IJN.S277352

Single anonymous peer review

Editor approved for publication: Dr. Thomas Webster

Fatma Mohamed Elsenosy, 1 Ghada Ahmed Abdelbary, 2 Ahmed Hassen Elshafeey, 2 Ibrahim Elsayed, 2, 3 Ahmed Roshdy Fares 2 1 Department of Clinical Pharmacy, Children's Cancer Hospital, Cairo 57357, Egypt; 2 Department of Pharmacy and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt ; 3 Department of Pharmacy, Ajman Gulf Medical University and Thumbay Institute of Precision Medicine, United Arab Emirates 11562, Egypt Tel 20 12 88285866 Email [email protected] Purpose: Duloxetine (DLX) is a dual serotonin and Norepinephrine reuptake inhibitors have limited bioavailability (≈ 40%) due to extensive liver metabolism. This work aims to develop and evaluate the DLX intranasal thermoreversible cube gel to improve its bioavailability and ensure effective brain targeting. Materials and methods: Cubo-gel is prepared by a 33-center composite design with three independent factors, lipid ratio (glycerol monooleate: glycerol tripalmitate), Pluronic F127% and Pluronic F68%. The particle size (PS), gelation temperature (GT), encapsulation efficiency (EE%) and in vitro release of the prepared formulations were evaluated. Choose the cubic gel with the highest desirability (0.88) as the optimized formula. Differential scanning calorimetry, Fourier transform infrared spectroscopy, X-ray powder diffraction, and transmission electron microscopy were used to evaluate DLX cubic gels. Conducted cytotoxicity studies, in vitro penetration studies, and in vivo biodistribution studies to evaluate the safety and effectiveness of brain targeting. Results: The best cubic gel is composed of 3.76 lipid ratio, 20% w/v PF127 and 5% w/v PF68. Its PS is 265.13 ± 9.85 nm, GT is 32 ± 0.05°C, EE% is 98.13 ± 0.50%, and it exhibits a controlled release behavior, of which 33% DLX is released within 6 hours. Compared with DLX solution and DLX-loaded in-situ cubic gel, ordinary in-situ cubic gel has significantly higher IC50. In vitro permeation studies showed that the drug permeation from DLX in-situ cubic gel was enhanced by 1.27. According to the study of in vivo biodistribution in plasma and brain, the brain bioavailability of intranasal DLX in-situ cubic gel is 1.96 times higher than that of intranasal solution. Its BTE% and DTP% are 137.77 and 10.5, respectively, indicating that brain targeting is effective after intranasal administration. Conclusion: Therefore, intranasal DLX in-situ cubic gel can be considered as an innovative nano-carrier delivery system for enhancing the bioavailability of DLX and effective brain targeting to maximize its effects. Keywords: Duloxetine, central composite design, cube, thermoreversible in situ gel, intranasal, brain targeting

Duloxetine (DLX), {(3S)-N-methyl-3-naphthalene-1-acyloxy-3-thiophene-2-ylpropan-1-amine}, is a dual serotonin and norepinephrine re Intake inhibitors for the treatment of major depression. 1 It is also used to treat urinary incontinence, fibromyalgia and diabetic peripheral neuropathic pain. 2 DLX is a class II drug of the Biopharmaceutical Classification System (BCS) with limited water solubility. According to reports, the solubility of DLX is 2.68 mg/mL at pH 6.8.3. DLX is well absorbed after oral administration, but has a lag time of 2 hours before the start of absorption, and reaches its tmax 6 hours after administration. Due to extensive liver metabolism, the bioavailability of DLX is limited and variable (40% to 80%), and it is also degraded in acidic gastric media, resulting in lower than therapeutic levels. 4,5

Intranasal administration is an advanced route of administration, which can easily self-administer without sterile equipment. 6 Since the drug is absorbed through a large single epithelial layer, the drug can quickly enter the systemic circulation from the nasal cavity. 7 Nasal administration has an advantage in the brain to directly transfer drugs to the brain through neurons and extracellular routes and avoid the blood-brain barrier (BBB) ​​for targeting. 8 The most significant advantage of nasal administration is that it bypasses first-pass liver metabolism, has low enzyme activity, and is directly transported to the systemic circulation. In addition, nasal administration provides rapid onset, ease of administration, and non-invasiveness. On the other hand, the intranasal route has limitations. For example, the volume of the preparation that can be inserted into the nasal cavity is limited, the rapid flushing of mucociliary leads to short residence time, and the absorption of hydrophilicity and macromolecules is poor. These limitations can be overcome by preparing drug-loaded in-situ cubic gels.

New nanovesicle systems such as liposomes, cubes and niosomes are considered interesting candidates for nose-to-brain drug delivery. Their size and physical properties make them a new promising tool that can increase the residence time of the drug at the absorption site, protect the encapsulated drug from degradation, promote mucosal penetration, and control the release profile of the encapsulated drug. 9

The cube is one of the new nanocarriers. It consists of a bicontinuous lipid bilayer that separates two networks of water channels. The cube is composed of amphiphilic polar lipids, such as glycerol monooleate (GMO), which is the most common polar lipid used in cube formulations in the presence of stabilizers such as Pluronic F127 (PF127). 10 GMO can self-assemble in water to form a micelle structure when its concentration exceeds the critical micelle concentration (CMC). At higher concentrations, it will form a bicontinuous cubic structure. 11,12 Cubes have many advantages, such as high embedding efficiency, controlled drug release, biocompatibility, bioadhesive properties, and thermodynamic stability. 13

In situ intranasal gel is a solution at room temperature (<25°C), which forms a gel when inserted into the nasal cavity (32°C to 34°C), which can prolong the residence time of the preparation in the nasal cavity, thereby increasing the permeability 14 in situ nasal cavity The gel is composed of thermosensitive polymers such as chitosan, pluronic, xyloglucan and hydroxypropyl methylcellulose. 15 Pluronic F127 (PF127) and Pluronic F68 (PF68) are the most commonly used thermosensitive polymers. Pluronics is a water-soluble triblock copolymer composed of polyethylene oxide (PEO) and polypropylene (PPO) parts. The ratio between PEO and PPO determines the hydrophobicity and hydrophilicity, respectively. 12

Central composite design (CCD) is a practical statistical experimental design. Compared with full factorial design, it uses fewer experimental runs to study the main effects of experimental factors and their interactions. It covers many possible combinations, and only three levels are required for each factor. CCD can be used to predict and optimize the response to prepare the best formula. 16,17

In a previous study, Alam et al. examined the effect of intranasal infusion containing DLX nanostructured lipid carriers (NLC) on improving the amount of DLX in the brain and plasma. 18 In another study, Khatoon et al. evaluated intranasal administration of a thiolated chitosan gel containing DLX prosaccharide to improve its brain delivery. 19

This study aims to formulate and characterize DLX in-situ cube gel to improve its bioavailability and enhance its brain targeting. The three-factor, three-level CCD is used to study the influence of different variables on the research response to prepare an optimized formula. The independent variables selected are: lipid ratio [glycerol monooleate (GMO): glycerol tripalmitate (GTP)] (A), PF127 percentage (B), and PF68 percentage (C). The dependent variables selected in the study include: particle size (PS) [Y1], gelation temperature (GT) [Y2], retention efficiency percentage (EE%) [Y3] and release percentage after 6 hours (Q6) [Y4]. The best DLX in-situ cubic gel was physically characterized using a transmission electron microscope (TEM). Finally, in vitro penetration and in vivo biodistribution studies were performed to characterize the best formulation performance for brain targeting.

As far as we know, there has been no previous study on the preparation of GTP-containing cubes, combined with GMO, to study its effect on the brain targeting ability of EE%, Q6 and cubes placed in the in-situ gel system. This new formulation is designed to combine the advantages of the in-situ gel system, that is, it is easy to instill in the nose, has a long retention time, and the drug is continuously released after being transformed into a gel; with the small particle size and lipophilicity of the cube, it can achieve better DLX Permeability and brain targeting.

Duloxetine hydrochloride (DLX) was kindly provided by EVA Pharma in Cairo, Egypt. Pluronic F127 (PF127), Pluronic F68 (PF68), glyceryl monooleate (GMO) and glyceryl tripalmitate (GTP) were purchased from Sigma-Aldrich, St. Louis, USA. All other chemicals and solvents are of analytical grade and can be used without further purification.

Use Design-Expert® software (version 7, Stat-Ease Inc., Minnesota, USA). The independent variables are: lipid ratio [GMO ratio: GTP] (A), PF127% (B) and PF68% (C). The level of the factor is selected as (-1, 0, and 1). The composition of the formula and its actual representative values ​​are shown in Table 1. The dependent variables are particle size (PS) [Y1], gelation temperature (GT) [Y2], encapsulation efficiency (EE%) [Y3] and percentage released after 6 hours (Q6) [Y4]. The polydispersity index (PDI) and zeta potential (ZP) of the prepared formulations were also measured. The desirability value is calculated based on the response surface analysis of the obtained data. They are used to select optimized compositions with as many desired characteristics as possible. Table 1 Composition of 33 CCD used in DLX in-situ Cubo-Gels

Table 1 Composition of 33 CCD used in DLX in-situ Cubo-Gels

According to the above design, according to the cold method previously described by Soga et al. and slightly modified, different Pluronic solutions were prepared. 20 PF127 and PF68 are accurately weighed and dissolved in 10 mL of distilled water with continuous stirring at 1000 rpm. The dispersion was hydrated overnight at 4°C to obtain a homogeneous glassy solution. Table 1 shows the composition of the different gelling systems prepared.

Accurately weigh different proportions of GMO and GTP and mix them with 50 mg DLX. The lipid mixture was melted in a 70°C water bath to obtain a clear lipid melt. The different ratios of GMO and GTP are shown in Table 1. At 1000 rpm and 25°C, the homogeneous lipid melt was added dropwise to the magnetically stirred Pluronic solution and left for 1 hour.

The dynamic light scattering technique is used to analyze the cube PS (Zetasizer Nano ZS-90, Malvern Instruments and Worcestershire, UK). Before measurement, take 1 mL of each preparation and dilute it with distilled water to translucent. In addition, the PS distribution is evaluated by measuring PDI. Finally, the physical stability was evaluated by analyzing the ZP of the diluted formulation sample. Three repeated measurements are provided for three separate samples of each formula; the average value of each ± ​​standard deviation (SD) is determined.

GT is measured using the tilt method described by Zaki et al. 21 Transfer a 2 mL aliquot of the prepared formulation to a test tube and immerse it in a water bath. The temperature of the water bath was gradually increased by 1 degree and equilibrated for 5 minutes at each new temperature. Then check the gel for the sample. When the test tube is tilted at an angle of 90° and the meniscus of the sample no longer moves, confirm gelation.

EE% is a measure of the content of DLX enclosed in a cube. Evaluate EE% using ultrafiltration method. Filter a 0.5 mL aliquot from each formulation using a 0.22 μm syringe filter (nylon 25 mm Luer syringe filter), which is equivalent to 2.5 mg DLX. After filtration, the clear filtrate was diluted with distilled water, and the content of free DLX was analyzed at λmax 286 with an ultraviolet spectrophotometer. EE% is calculated as follows: 22 (1)

Where Ct is total DLX and Cf is free DLX.

The membrane diffusion method was used to evaluate the in vitro release of DLX. 23 Transfer 1 mL aliquots of each formulation equivalent to 5 mg DLX to the release medium (12-14 kDa, Sigma-Aldrich, St. Louis, USA). Then immerse the dialysis membrane in 45 mL of phosphate buffered saline (PBS) pH 7.4 (to maintain tank conditions) and keep it at 37 ± 0.5ºC. The medium is stirred at 50 rpm. At specified time intervals (0.5, 1, 2, 3, 4, 6 hours), take 3 mL samples and immediately replace them with the same volume of fresh medium. The sample taken out was analyzed by an ultraviolet spectrophotometer at λmax 286 nm.

Use Design Expert® software to select the best formula, where the minimum PS, GT and Q6 are related to the maximum EE%. After that, an optimized formula is prepared and the same technique is used for physical characterization to verify the result using a predetermined technique. Finally, the optimized formula was prepared and freeze-dried at a condenser temperature of -45ºC for 24 hours (Alpha 1-2 LD plus CHRIST, Germany) for more characterization.

The in vitro release of PS, PDI, ZP, GT, EE% and optimized formulations were measured using the same methods and techniques previously used to characterize the initial formulation set.

The gel time is the time required for the formulation to turn from solution to gel when inserted into the nasal cavity. Transfer an aliquot of the 1mL formulation to a test tube, and then install it horizontally in a water bath adjusted to 33±0.5°C. Use a stopwatch to record the time required to form the gel. twenty four

Dilute a 1 mL aliquot of the optimized formula with 10 mL distilled water, and then measure the pH of the resulting solution with a pH meter (Beckman Coulter, USA), which was previously calibrated with pH 4 and pH 7.25 buffers

Use TEM (JXA-840; JEOL, Tokyo, Japan) to photograph the shape, shape and size of the best cubic gel. The cube gel was applied to the carbon-coated copper array, and then the cube was inspected by HR-TEM in bright field mode at a working voltage of 200 kV.

DSC was performed on the in-situ cubic gel and free DLX loaded by DLX to detect any physical changes after the DLX was wrapped in the cube, and used (DSC-60, Shimadzu, Japan) to analyze its compatibility. Place the sample (3–4 mg) in an aluminum pan and heat it at a constant rate of 10°C/min. Using the empty pan as a reference, heat to a temperature range of 30°C-350°C in a nitrogen atmosphere.

FTIR (Thermo Scientific Nicolet 6700, USA) was used to analyze DLX and drug-loaded in-situ cubic gel to detect any possible interactions. The sample is compressed into a disc with KBr. Scan the sample in the range of 400 to 4000 cm-1.

Use an X-ray diffractometer (model XD-610, Shimadzu, Kyoto, Japan) with Cu as the tube node to perform the X-ray diffraction pattern of free DLX and optimized in-situ cubic gel. Record the diffraction pattern under the following conditions: voltage 45 kV, current 30 mA, step length 0.02°, counting rate 0.5 s/step, room temperature. Use a scattering angle (2θ) ranging from 4° to 50°. 10 to collect data

The study was conducted on oral epithelial cells purchased from Nawah Scientific Inc. in Cairo, Egypt. The cells are supported by Dulbecco's modified Eagle's medium containing penicillin, streptomycin, and fetal bovine serum at concentrations of 100 U/mL, 100 mg/mL, and 10%. The 26 cell suspension (100 μL aliquot) was incubated in a 96-well plate under carbon dioxide for 24 hours at 37°C. Add 100 μL of DLX solution samples of different concentrations (0.01, 0.1, 1, 10, 100 μg/mL), optimized pure in-situ cubic gel and optimized DLX-loaded in-situ cubic gel to the cell suspension. After 72 hours, an aqueous solution of trichloroacetic acid (150 μL, 10% w/v) was added to the cells as a fixative. After incubating for 1 h at 4°C, wash the cells 5 times with distilled water, mix with 70 μL Sulforhodamine B solution (0.4% w/v), and incubate again at 25°C in the dark for 10 minutes. The 27 cells were washed 3 times with 1% w/v acetic acid solution, dried overnight, and then mixed with TRIS buffer (150 μL, 10 mM). Finally, use the BMG LABTECH®-FLUOstar Omega microplate reader (Ortenberg, Germany) to measure the absorbance at 540 nm.

In vitro penetration studies were used to determine the ability of the cube to improve drug penetration through the nasal mucosa and effective brain targeting. Compare the DLX penetration from the optimized cubic gel with the DLX solution. The nasal membranes were taken from the nostrils of Rahmani sheep, with an average age of 6 months and a weight of 32 kg. 22 The intact membrane is identified, separated, cleaned and stored frozen. The cut film (1 cm diameter) is fixed to one end of a specially designed glass tube. Fix the tube vertically from the other end in a glass container containing 50 mL PBS (pH 7.4). Add a 1 mL aliquot of the best formula equivalent to 5 mg DLX into the glass tube. Immerse the glass container in a horizontal shaking water bath at a temperature of 37°C ± 0.5 and 50 rpm for 24 hours. Take 3 mL samples at different time intervals (0.5, 1, 2, 3, 4, 6 and 24 hours) and replace the volume lost due to sample withdrawal with fresh medium. The samples were analyzed by HPLC (Shimadzu, Tokyo, Japan). The mobile phase consisted of acetonitrile in 10 mM phosphate buffer pH 4.5 in a ratio of 55:45. The flow rate is 1 mL/min. DLX is detected by an ultraviolet detector (SPD-10 A, Shimadzu, Tokyo, Japan) at a wavelength of 288 nm. 5 All measurements are performed in triplicate, and the average value ± SD is reported. The amount of drug penetrating is calculated as a function of the surface area of ​​the nasal membrane (μg/cm2). Then, calculate the 24-hour drug flux (J24) by the following formula: 22,28 (2)

The drug permeates from the optimized formula, and the drug solution is statistically compared by one-way analysis of variance, and then passed the Fisher least significant difference test. The enhancement ratio (ER) was calculated to evaluate the efficiency of the cubic gel to improve permeability compared to the drug solution. ER is calculated using the following formula: 22,29 (3)

The animal experiment research protocol was approved by the experimental and clinical research ethics committee of the Faculty of Pharmacy of Cairo University [PI (2194)]. The research follows the principles of the ethical guidelines outlined by the International Committee for Laboratory Animal Science (ICLAS). 30,31 Swiss albino rats (number: 46, average weight: 100 grams) were randomly divided into four groups. The first group and the second group were given the optimized in-situ cubic gel formulation and drug solution via intranasal (IN) route, respectively. The third group was given the intravenous (IV) formulation with the best cubic gel of the same composition, except that the PF127% was reduced to 10% to avoid gel formation after the IV formulation. Finally, the fourth group injected the drug solution intravenously. The following formula is used to calculate the animal dose: 32 (4)

Km is the conversion factor, which is equal to 37 and 6 for humans and rats, respectively.

The optimized formula and drug solution used are equivalent to 5 mg DLX. Polyethylene tube (inner diameter: 0.1 mm), installed on a Hamilton syringe, for nasal administration. IV administration via tail vein. At each time interval (0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 24, 48, and 72 hours), four rats were anesthetized and sacrificed, one in each group. Separate the brain from each rat and collect blood samples. The clear plasma is separated by centrifuging the blood sample at 4000 rpm for 15 minutes. Plasma samples are stored in a refrigerator at -80°C until analysis. Each separated brain was washed with normal saline (NS), weighed, and homogenized in PBS (pH 7.4) using a homogenizer (Heidolph DIAX 900, USA) to form a 50% homogenate. The sample is kept at -80°C until analysis.

For the preparation of the standard calibration curve, a defined volume of DLX stock solution and 25 µL reboxetine solution (5 mg/mL) are used as the internal standard (IS); it is added to 0.5 mL of plasma to construct a calibration standard with the following concentrations : 0.1, 0.2, 1, 5, 20, 40, 60, 80, 100, 150, 200 and 250 ng/mL. For sample preparation, add 25 µL of internal standard to 0.5 mL of sample (plasma or brain homogenate) and vortex in a 10 mL glass tube for 1 minute. Then, as a protein precipitant, 4 mL of ethyl acetate was added to the previous sample, vortexed for 3 minutes, and then centrifuged at 3000 rpm for 10 minutes. The organic layer (3 mL) was separated into a new tube and dried under vacuum using a centrifugal vacuum concentrator (Eppendorf 5301; Hamburg, Germany) at 45°C. The dry residue was diluted in 500 µl of mobile phase, and (20 µL) of the solution was injected into the liquid chromatograph and analyzed by tandem mass spectrometry (LC-MS/MS). 33

Analyze plasma and brain samples using an LC-MS/MS (Shimadzu, Japan) system equipped with a degasser (DGU-20A3), solvent distribution device (LC-20AD) and autosampler (SIL-20 A). A sensitive and validated LC-MS/MS system was established for the quantitative analysis of DLX using reboxetine as an internal standard. The detection of MS/MS was carried out in positive ion mode at 550°C using AB Sciex API-3200 mass spectrometer (Foster, California, USA) equipped with turbo ion spray interface. The ion spray voltage is adjusted to 5500V. For analysis, an aliquot of 25 µL of the analytical sample was injected onto a C18 chromatographic column with 100 A (50 x 4.6 mm) (Phenomenex, USA), PS 5 µm. The mobile phase is isocratic and consists of an aqueous solution of acetonitrile and 0.5% formic acid in a ratio of 80:20 (v/v). Adjust the flow rate to 1 mL/min. Use multiple reaction monitoring (MRM) mode for ion detection. In DLX, the transition from m/z 298.1 precursor ion to m/z 154.2; for reboxetine internal standard, it is from m/z 314.2 precursor ion to m/z 175.1. The data has been analyzed using version 1.4.2 analysis software (Applied Biosystems Inc., Foster City, USA). 33

The pharmacokinetic characteristics of DLX were described using a non-compartmental model after the optimal formulation and drug solution were administered to rats with IN. The maximum concentration (Cmax) and peak time (tmax) are obtained directly from the concentration-time curves of plasma and brain. Other pharmacokinetic parameters, such as elimination rate constant (Kel), elimination half-life (t1/2), mean residence time (MRT), zero to last (AUC0-72) and infinity (AUC0-inf.) Use Kinetica Software version 5 (Thermo Fisher Scientific, MA, USA) calculation. Estimate the brain targeting efficiency by determining the brain targeting efficiency (BTE%) and the drug delivery percentage (DTP%). BTE% is the brain exposure to the drug after IN administration compared with IV administration. Calculated by the following formula: 9,34 (5)

BIN and PIN are AUC0-inf. Respectively in brain homogenate (B) and plasma (P), after optimizing the formulation and the IN administration of the drug solution. On the other hand, BIV and PIV are AUC0-inf. After IV administration, in brain homogenate (B) and plasma (P). The BTE% of the value ranges from 0 to ∞. Compared with IV, the effective brain targeting after IN administration showed a value above 100%. 9

In addition, DTP% is the ratio of the drug transferred directly from the nose to the brain from the IN dose to the total amount of drug that reaches the brain after the IN is delivered. The calculation formula is as follows: 9,35 (6) (7)

A positive DTP% value of up to 100% indicates that the brain drug level is largely due to the transfer directly from the nose to the brain, while a DTP% equal to 0 (or even negative) indicates that the main route for the drug to enter the brain is after intravenous administration Systemic circulation. 9

The stability study of DLX in-situ cubic gel was carried out to ensure its physical stability. Take a 5 mL aliquot from the formulation and store it in a closed amber glass vial at room temperature (25°C) and refrigerator (4°C) for 3 months. After 3 months, evaluate the sample by measuring its PS, PDI, ZP and EE%. All the data obtained are the average ± SD of three measurements.

All data are expressed as mean ± standard deviation (SD). The Design-Expert® software (version 7, Stat-Ease Inc., MN, USA) was used to generate 33 CCDs and perform statistical analysis. Analysis of variance (ANOVA) is used to prove the significance of each factor. Statistical analysis of penetration studies and pharmacokinetic parameters was performed by SPSS 17.0 software (SPSS Inc., Chicago, USA) using one-way analysis of variance test. If the p-value is <0.05, then the difference between the averages is considered significant.

The PS of the cube must be designed to be 100 to 300 nm in order to achieve brain targeting. 13 The PS range of the prepared cube is 145.5 nm to 515.9 nm, as shown in Table 2. This finding is within the acceptable range of brain targeting. 13 In a previous study, the PS range of proniosomes for brain-targeting DLX prepared by Khatoon et al. was 166 to 842 nm. 19 The smallest size belongs to F4/v PF68 prepared with a lipid ratio of 0.1, 20% w/v PF127 and 5% w. The linear model is the preferred model for PS. The difference between the adjusted R2 and the predicted R2 must be less than 0.2 to show how the model predicts the response. The PS model shows adjusted R2 (0.6613) and predicted R2 (0.538). The calculation formula for PS analysis is: Table 2 Average PS, GT, EE%, Q6, PDI and ZP of prepared DLX in-situ Cubo-Gels

Table 2 Average PS, GT, EE%, Q6, PDI and ZP of the prepared DLX in-situ cubic gel

PS = 324.94 77.93 A-42.2B 34.18C (8)

The effect of lipid ratio (A) and PF127% (B) on PS is shown in Figure 1A. The analysis of variance showed that the lipid ratio and PF127% had a significant effect on PS (p <0.05). Increasing lipid ratio, decreasing PF127% and increasing PF68% resulted in greater PS. PF127 is a polymer surfactant that can be used as a stabilizer for cubes. Increasing the concentration of PF127 stabilizes the system spatially and effectively distributes the crystal structure of the cube. 36,37 In addition, increasing PF127 reduces the surface tension, which helps to form a cube with a smaller PS. In a previous study, Abdelrahman, FE, and others reported that increasing the concentration of PF127 resulted in a decrease in the PS of cubes made of risperidone. 22 In another study, Khatoon et al. reported that increasing the concentration of Tween 80 resulted in a decrease in the PS of DLX due to a decrease in surface tension, resulting in proglycosome. 19 GMO is an amphiphilic polar lipid. When the concentration is higher than CMC, it will form micelles. At higher concentrations, it will form cubes. 13 Increasing the lipid ratio means increasing the GMO and increasing the PS of the formed cube. Figure 1 3D surface diagram of the main effects and interactions of lipid ratio, PF127 percentage and PF68 percentage on (A) PS (B) GT, (C and D) EE% and (E) Q6. Lipid ratio has a significant effect on PS, EE% and Q6; PF127% has a significant effect on PS, GT and EE%; PF68% has a significant effect on EE% and Q6.

Figure 1 The 3D surface diagram of the main effects and interactions of lipid ratio, PF127 percentage and PF68 percentage on (A) PS (B) GT, (C and D) EE% and (E) Q6. Lipid ratio has a significant effect on PS, EE% and Q6; PF127% has a significant effect on PS, GT and EE%; PF68% has a significant effect on EE% and Q6.

PDI is an indicator of the uniformity of PS distribution. 38 A lower value indicates monodispersity, while a higher value indicates polydispersity. The PDI results range from 0.3 to 1, as described in Table 2. The lowest PDI value of F9 is 0.222 ± 0.02.

ZP is a stability indicator. It reflects the aggregation tendency of nanoparticles. The larger the ZP, the greater the repulsive force that reduces particle aggregation. 39 The ZP range of the prepared cube is 1.6 to 10.6 mv, as shown in Table 2. PF127 has a negative effect on ZP because of its non-ionic nature. On the other hand, PF127 can act as a steric stabilizer to prevent aggregation of the prepared cubes. 12,40,41

The thermoreversible in-situ nasal gel is a free-flowing liquid at room temperature and will turn into a gel when inserted into the nasal cavity. 15 The temperature at which the solution turns into a gel is called the gel temperature (GT). 42 The GT range of the design formula is 32°C to 80°C, as shown in Table 2. The range of 33°C-35°C is most suitable for gel in the nasal cavity. 43 The lowest GT (32°C ± 1) belongs to F14, which contains 20% PF127 and 5% PF68. The quadratic model is the most suitable model for polynomial data analysis (p<0.001). The predicted R2 (0.9822) is consistent with the adjusted R2 (0.9550). The equation used to describe GT is:

GT = 46.55 0.1A-22.7B 0.7C-0.12AB 0.38AC 0.88 BC 2.14 A2 8.14B2 1.14 C2 (9)

Analysis of variance showed that GT only depends on PF127%, p<0.001. PF127 has a negative effect on GT; increasing PF127% will result in a significant decrease in GT. The effect of PF127% on GT is shown in Figure 1B. Pluronics is a linear triblock copolymer consisting of a polypropylene block (PPO) between two polyethylene oxide blocks (PEO). The amphipathic properties depend on the length of PPO and PEO. Pluronic can self-assemble into micelles with a hydrophobic PPO core and a hydrophilic PEO shell. By increasing the concentration, gelation occurs at a lower temperature due to the accumulation of micelles. PF127 and PF68 are the most commonly used polymers in thermoreversible gels. PF127 forms a gel at a lower temperature than PF68. Data analysis shows that the formulation containing 20% ​​PF127 forms a gel at a lower temperature. Since the hydrophilicity of PF68 will destroy the hydration shell around PF127.24, adding PF68 to PF127 will further reduce the sol-gel transition temperature

As shown in Table 2, the obtained results show that the EE% is between 93.1% and 99.7%, indicating that the prepared cubes show a higher drug encapsulation efficiency. Using a two-factor interaction model, there is an acceptable difference between the adjusted R2 (0.96) and the predicted R2 (0.76). The equation describing EE% is:

EE% = 98.16 0.85A 1.13B 0.81C-0.66AB-0.68AC-0.60BC (10)

Statistical analysis of variance showed that all variables had a significant positive effect on EE% (p<0.001). Figure 1C and D show the effect of lipid ratio, PF127% and PF68% on EE%. GMO is an amphiphilic polar lipid. It can self-assemble into a bi-continuous cubic structure in water. 12 The cubic structure formed has strong encapsulation ability for different drugs with different molecular weights and polarities. 44 The second factor affecting EE% is PF127%. PF127 is a water-soluble non-ionic triblock copolymer with HLB=22.45. PF127 has multiple functions in this design; it acts as a solubilizer for drugs, a stabilizer for cubes, and a gelling agent. PF127 can increase the water solubility of poorly soluble drugs and increase the retention of drugs in the cube water channels. 46 In addition, PF127 can stabilize the drug-trapping cube by forming a coating on it. The jacket can retain excess DLX to increase its retention. 37 The last factor affecting the retention is PF68%. Like PF1​​27, PF68 is a water-soluble copolymer with solubilization efficiency, but lower than PF127, so it can improve water solubility and encapsulate poorly soluble drugs such as DLX. 46 In previous studies, it was pointed out that increasing the concentration of Tween 80 increased the EE% of DLX in the prepared precursor due to increased drug wettability. 19 In another study, it was reported that increasing the concentration of Tween 80 increased the EE% of risperidone because of the extra amount of drug retained in the prepared cube. twenty two

Figure 2 shows the DLX release of different cubic gel formulations, and Table 2 shows the Q6 value. These formulations showed slow drug release. The range of DLX release after 24 hours was from 42.77 ± 7.01 to 101.38 ± 1.7%. In a previous study, it was reported that the prepared precursor gel and mucoadhesive precursor gel released 30% and 24% of DLX after 24 hours at pH 7.4. 19 This shows that our DLX cubic gel can provide better controlled release profile and greater DLX degree compared with the formulations in the previous study. Polynomial analysis, combined with a two-factor interaction model, is used to illustrate the data released in Q6. There is an acceptable difference between the adjusted R2 (0.908) and the predicted R2 (0.729). Analysis of variance showed that lipid ratio and PF68% were factors that significantly affected Q6 (p<0.05). Figure 1E shows the effect of lipid ratio and PF68% on Q6. The equation describing Q6 is: Figure 2 In vitro release curve of DLX in-situ cubic gel in PBS (pH = 7.4) at 37°C. The range of Q6 is 20.3±2.14 to 64.5±4.13%, and the range of release degree is 42.77±7.01 to 101.38±1.7%.

Figure 2 The in vitro release curve of DLX in-situ cubic gel in PBS (pH = 7.4) at 37°C. The range of Q6 is 20.3±2.14 to 64.5±4.13%, and the range of release degree is 42.77±7.01 to 101.38±1.7%.

Q6 = 35.63–3.24A 0.74B 2.38C-6.54AB-5.32AC 10.13BC (11)

The lipid ratio has a negative effect on the released Q6. GMO is the most common lipid in cube formulations; it may reduce the release of the drug by delaying the distribution of the drug from the oily medium to the aqueous medium. 22 GTP is a hydrophobic triglyceride formed by the acylation of three hydroxyl groups of glycerol with palmitic acid. It will reduce the wettability of the formulation and the release medium, thereby reducing the release of the drug. 47 On the other hand, PF68% has a positive effect on Q6; increasing PF68% results in a significant increase in the drug released from the cube. PF68 is an amphiphilic copolymer and can be used as a pore former and release accelerator. 48

The optimized formula with an ideality value of 0.88 (consisting of 3.76 lipid ratio, 20% w/v PF127 and 5% w/v PF68) has a PS of 265.13 ± 9.85 nm, a PDI of 0.42 ± 0.04, and a ZP of 2.79 ± 0. millivolt. GT is 32±0.05°C. EE% is 98.13 ± 0.50%, Q6 is 33%. These findings are consistent with the values ​​predicted by the statistical design performed.

It is the time it takes for a thermally responsive formulation to form a gel (sol-to-gel transition) at a specific temperature. When kept at 33 ± 0.5°C, the cubic gel turns into a gel after 10 seconds.

The pH of the intranasal product must be measured to avoid irritation of the nasal mucosa. The pH of the intranasal preparation must be 5 to 6.5.49. The pH of the cubic gel is 6.3. This result is consistent with the results of intranasal delivery of DLX reported by Khatoon et al. They reported that the pH values ​​of the DLX precursor gel and mucoadhesive precursor gel were 6.44 and 5.67, respectively. 19

Observe that the shape of the cube is not a typical cube, as shown in Figure 3. In previous studies, Nasr et al. and Abo El-Enin et al. found that the drug-loaded cubes are almost spherical and have irregular polygonal shapes. 10, 37 This may be due to the semi-cubic (hemispherical) shape caused by GTP. As reported in previous studies, GTP forms dense spherical nanoparticles. 50,51 The scanned cube shows many water channels in its structure. For example, GMO self-assembles in water to form a liquid crystal cubic phase, which is composed of a bicontinuous lipid bilayer with a network of water channels. 10 The cubes are well dispersed without agglomeration; their surface is smooth and irregular. Their size is consistent with the size of the PS result. This PS is suitable for brain targeting through BBB. Figure 3 (A) Optimized DLX in-situ cube gel formulation and (B) enlarged TEM image of a single cube. The image shows a cube with many water channels in its structure.

Figure 3 (A) Optimized DLX in-situ cube gel formulation and (B) enlarged TEM image of a single cube. The image shows a cube with many water channels in its structure.

The DSC thermogram of DLX and optimized in-situ cubic gel is shown in Figure 4. The DLX thermogram showed a high endothermic peak at 167.87°C, indicating the crystalline state of the drug. 3 However, the lyophilized cube has no sharp peaks, but has two small peaks at 51.4°C and 65°C. This may indicate that when DLX is formulated into a cube gel, it is wrapped in a cube in an amorphous state, or an interaction may have occurred. Figure 4 The DSC thermogram of DLX shows the endothermic peak at 167.87°C, while the DSC thermogram of the best DLX in-situ cubic gel shows the disappearance of the DLX endothermic peak.

Figure 4 The DSC thermogram of DLX shows the endothermic peak at 167.87°C, while the DSC thermogram of the best DLX in-situ cubic gel shows the disappearance of the DLX endothermic peak.

The FTIR spectra of DLX and lyophilized best in-situ cubic gels are shown in Figure 5. The FTIR spectra of GMO, 52, 53 GTP, 50, 51 PF127, 52, 54 and F6855 were obtained from previous studies found in the literature. The DLX spectrum shows clear peaks at 56 points at 1577.77 (aromatic olefin), 1462.04 (thiophene ring) and 1234.44 (carbonyl). 56 The spectrum of the lyophilized preparation showed that the peak intensities of these groups shifted and decreased slightly. These findings are consistent with their DSC thermograms, and may be due to the possible interaction between the GMO hydroxyl group and the carbonyl group in the drug. 57 Figure 5 The FTIR spectra of DLX and the best DLX in-situ cubic gel are shown at 1577.77 (aromatic olefin), 1462.04 (thiophene ring) and 1234.44 (carbonyl).

Figure 5 FTIR spectra of DLX and the best DLX in-situ cubic gel show that the peaks at 1577.77 (aromatic olefin), 1462.04 (thiophene ring) and 1234.44 (carbonyl) have shifted and decreased.

Figure 6 shows the XR diffraction patterns of DLX and lyophilized optimized DLX loaded in-situ cubic gels. The DLX diffraction pattern shows obvious peaks at 18.2°, 19.07°, 21.08°, 23.5° and 28.1° (2θ), and the relative intensities are 45.32, 60.06, 100, 50.43 and 38.08, respectively, which indicates that the crystal form has disappeared. In the diffractogram of a cubic gel loaded with DLX. This may indicate that the molecules of the drug are dispersed within the cube, which results in the loss of its crystalline properties, and may indicate that the drug is completely encapsulated in the cube. Figure 6 The XRPD of DLX showed obvious peaks at 18.2°, 19.07°, 21.08°, 23.5° and 28.1°, while the XRPD of the best DLX in-situ cubic gel showed the disappearance of these peaks.

Figure 6 The XRPD of DLX showed obvious peaks at 18.2°, 19.07°, 21.08°, 23.5° and 28.1°, while the XRPD of the best DLX in-situ cubic gel showed the disappearance of these peaks.

Carry out cytotoxicity test to ensure the safety of the cubic gel components to epithelial cells. As shown in Figure 7, compared with the drug solution and drug-loaded in-situ cubic gel, the best pure in-situ cubic gel has a significantly higher IC50 (70.85 μg/mL), and its IC50 values ​​are almost similar ( 21.66 and 20.77 μg/mL respectively). The results show that compared with drugs, the safety of the ingredients of the formula is increased by 3.27 times. In addition, the formulation components have no synergistic effect on the cytotoxicity of the drug. Ali-Boucetta et al. and Desai et al. studied the safety of PF127 and lipids, respectively. 58,59 Figure 7 The cell viability of the optimized DLX in-situ cubic gel compared with the ordinary in-situ cubic gel and the DLX solution on the oral epithelial cells showed that they were almost similar to the DLX solution with almost similar IC50 values ​​(21.66 and 20.77) Compared with DLX in-situ cubic gels, ordinary in-situ cubic gels have significantly higher IC50 μg/ml, respectively).

Figure 7 The cell viability of the optimized DLX in situ gel compared with the ordinary in situ gel and DLX solution on oral epithelial cells shows that the ordinary in situ gel has a significantly higher IC50 (70.85 μg/mL) DLX solution Its IC50 values ​​are almost similar to those of DLX in-situ cubic gels (21.66 and 20.77 μg/mL, respectively).

Permeation studies were used to evaluate the effect of cubic gel on the diffusion of DLX through the nasal membrane. This helps predict drug permeability in the body. Figure 8 shows the permeation curve of the loaded DLX in-situ cubic gel compared with the DLX solution. The drug fluxes (J24) of DLX in-situ cubic gel and DLX solution were 110.75 and 87.2 μg/h/cm2, respectively. This indicates a significant increase in permeability, p<0.001. The enhancement ratio was 1.27, indicating that the amount of drug permeated by the cubic gel per unit area of ​​the amniotic nasal membrane was increased compared with the DLX solution. A previous study reported that the fluxes of IN DLX in situ gel and mucoadhesive in situ gel were 8.6 and 16.1 μg/h/cm2, respectively. 19 Our results show that DLX in situ cubic gel The flux is 110.75 μg/h/cm2. This confirms the superiority of our formula in enhancing the penetration of DLX through the nasal mucosa compared with previous studies. Figure 8 Compared with the DLX solution, the cumulative amount of DLX permeated through the amniotic nasal membrane per unit area through the optimized DLX in-situ cubic gel has an enhancement rate of 1.27 compared to the increased permeability.

Figure 8 Compared with the DLX solution, the cumulative amount of DLX permeated through the amniotic nasal membrane per unit area through the optimized DLX in-situ cubic gel has an enhancement rate of 1.27 compared to the increased permeability.

The physical properties of Cubosomes and the components involved in their structure may be the main reason for the increased permeability. Nano-scale drug delivery systems ranging from 1 to 1000 nm can enhance mucosal penetration and cell internalization, while PS below 500 nm helps nanoparticles squeeze into the non-sticky water pores in the mucin network. 9 In a previous study, Shilo et al. found that the intracellular uptake of gold nanoparticles (GNP) strongly depends on GNP PS. They stated that when the drug is encapsulated in GNP, the optimal size to cross the BBB and brain cells is 70 nm. 60 In another study, Gao et al. showed that PS significantly affected the delivery of methotrexate nanoparticles across the BBB. They pointed out that when PS is lower than 100 nm, a significant difference in penetration of BBB is found, but nanoparticles of 100 to 400 nm do not significantly overcome BBB. 61

In addition, PS affects the cellular uptake and internalization of the prepared cubes. Bourganis et al. reported that nanoparticles smaller in diameter than olfactory axons (100 to 700 nm in human PS) can be transported to the brain in cells through olfactory nerve pathways. 62 Acosta concluded that nanoparticles with a PS smaller than 500 nm have higher cellular uptake than nanoparticles with a larger PS.63

In terms of composition, PF127 and PF68 are copolymer surfactants with enhanced penetration ability, so they can improve mucosal penetration. GMO is a polar lipid and the main component of the cube. It can promote intercellular lipid disorder through the interaction between its hydroxyl group and the anionic oxygen at the polar head of the phospholipid membrane, thereby acting as a permeability enhancer. effect. 64 These findings are consistent. The results reported by Abdelrahman, FE, and others indicate that the nano-size in the risperidone cube and the presence of Tween 80 can enhance the penetration of the drug through the nasal mucosa. twenty two

The calibration curve of DLX shows a linear response of 0.1–250 ng/mL with a coefficient of determination equal to 0.999. The analytical methods used in biodistribution studies have been validated to ensure their accuracy and precision. After intranasal (IN) and intravenous (IV) administration of preparations and drug solutions, the pharmacokinetics of DLX in plasma and brain homogenate were studied. The in vivo drug behavior in plasma and brain homogenate is shown in Figures 9 and 10. The pharmacokinetic parameters are shown in Table 3. Table 3 Pharmacokinetic parameters of DLX in plasma and brain homogenate after intranasal and intravenous administration optimized in situ Cubo Gel and drug solution Figure 9 Compared with IN solution, IV formulation and IV solution, IN DLX The mean plasma concentration-time curve of in-situ cubic gel in Swiss albino rats. Compared with the IN solution, the in-situ cubic gel showed a higher AUC0-inf, and the relative bioavailability was 188.92%. Figure 10 The average brain homogenate concentration-time curve of IN DLX in-situ cube gel compared with IN solution, IV formula and IV solution after administration of Swiss albino rats. Compared with the IN solution, the in-situ cubic gel showed a higher AUC0-inf, and the relative bioavailability was 196.13%.

Table 3 Optimized pharmacokinetic parameters of DLX in plasma and brain homogenate after intranasal and intravenous administration of Cubo-Gel and drug solutions in situ

Figure 9 The average plasma concentration-time curve of IN DLX in-situ cube gel and IN solution, IV formula and IV solution in Swiss albino rats after administration. Compared with the IN solution, the in-situ cubic gel showed a higher AUC0-inf, and the relative bioavailability was 188.92%.

Figure 10 The average brain homogenate concentration-time curve of IN DLX in-situ cube gel compared with IN solution, IV formula and IV solution after administration of Swiss albino rats. Compared with the IN solution, the in-situ cubic gel showed a higher AUC0-inf, and the relative bioavailability was 196.13%.

The pharmacokinetic results showed that the DLX level reached its peak concentration in plasma and brain after 0.15 hours of IN administration of cubo-gel preparation and drug solution. This short tmax may be due to rapid absorption via the IN route, while oral administration of DLX has a lag time of 2 hours before absorption, and the drug reaches its tmax after 6 hours. The plasma Cmax of the 65 IN cubic gel is 215 ± 7 ng/mL, and the IN solution is 239.63 ± 5.7 ng/mL. In brain tissue, the Cmax of the IN cubic gel is 51.8 ± 2.2 ng/mL, while the Cmax of the IN solution is 91.14 ± 4.15 ng/mL. In a previous study, Alam et al. prepared DLX nanostructured lipid carrier (NLC) and administered it as a circulating IN infusion to rats. DLX NLC showed that the drug concentration in the plasma was 186.9247 µg/mL, and the drug concentration in the brain was 228.88 µg/g. 18 Compared with our results, the higher absorption and permeability of IN DLX NLC may be due to many factors. First, the cyclic DLX NLC IN infusion covers a larger area of ​​the nasal mucosa and lasts longer, while DLX cubo-gel will be lost over time. Secondly, anesthetizing rats to promote the administration of DLX NLC IN infusion can reduce the nasal mucociliary clearance rate, thereby increasing the residence time and absorption of DLX NLC.

Although the Cmax of the IN solution in plasma and brain is higher than that of the cubic gel preparation, the in-situ cubic gel achieves a significant increase in the bioavailability of DLX, as shown by the higher values ​​of AUC0-72 and AUC0-∞. Shown in Table 3. Compared with IN solution, the relative bioavailability of cubo-gel in plasma and brain is 188.92% and 196.13%, respectively. This may be due to the controlled-release nature of IN cubo-gel and its higher residence time on the nasal mucosa, resulting in a lower Cmax at the beginning, but a higher final bioavailability.

The bioavailability of IN cubo-gel in the brain (208.43%) is also significantly higher than that of IV preparations. This may be due to the direct transfer of nasal preparations to the brain through the olfactory pathway and bypassing the BBB. On the other hand, IN cubo-gel is bioequivalent to IV solutions in plasma and brain, and bioequivalent to IV preparations in plasma. 66

In addition, the BTE% of IN cubo-gel (137.77%) is significantly higher than that of IN solution (75.02%), indicating that IN cubo-gel is more effective for brain targeting. This may be due to the fact that DLX directly penetrates into the brain through the olfactory pathway from the cubic gel or has a higher penetration rate through the BBB to some extent after being absorbed throughout the body. DTP% further supports these findings. The DTP% of the IN cube gel is 10.5%, but the DTP% of the IN solution is –13.7%. The positive DTP% in IN cubo-gel indicates that the direct nose-to-brain pathway is responsible for the high brain levels of DLX, while the negative DTP% in IN solution indicates that DLX preferentially passes through the systemic circulation and enters the brain after intravenous injection. 9

In addition, the higher bioavailability and advantages of IN cubo-gel in brain targeting compared to IN solution may be attributed to many other reasons. First, the instilled preparation turns into a gel when inserted into the nasal cavity, resulting in a longer residence time of the preparation on the nasal mucosa. This overcomes rapid mucociliary flushing, which is considered to be the most common problem of the IN route. 49,67 Second, the lipophilicity of the cube allows better vesicle penetration and more effective systemic absorption of the drug through the nasal mucosa and BBB. Absorb part of the formula. Third, Bourganis et al. reported the correlation between surface charge and DTP%. In particular, it can be observed that as ZP approaches zero, higher DTP% values ​​can be obtained. 62 Finally, according to BTE% and DTP%, a greater proportion of drugs reach the brain directly through the olfactory pathway, which is considered a unique pathway to the brain because it has no BBB protection. 9,68,69

The results in Table 4 show that the physical properties of the cube change very little. At room temperature, PS and EE% decrease slightly. In contrast, in a refrigerator (4-8°C), a slight increase in PS was found and a minimum decrease in EE%. The changes in ZP at room temperature and in the refrigerator are also very small. The small changes in PS, ZP and EE% were not statistically significant (P<0.05, paired t-test). The presence of high concentrations of PF127 and PF68 makes the system stable due to their amphiphilic nature. 45 Table 4 Average PS, ZP, PDI and EE% of Optimum DLX Cubo-Gel in situ at room temperature and refrigerator for 3 months

Table 4 Average PS, ZP, PDI and EE% of Optimum DLX in situ Cubo-Gel after 3 months of storage at room temperature and refrigerator

The thermoreversible DLX in-situ cubic gel was successfully prepared, with suitable GT, high EE%, small and uniform PS and controlled drug release. 33 CCD statistical analysis shows that lipid ratio has a significant effect on PS, EE% and Q6; PF127% has a significant effect on PS, GT and EE%; PF68% has a significant effect on EE% and Q6. The best DLX in-situ cubic gel with a desirability of 0.88 was prepared and evaluated. Its PS is 265.13 ± 9.85 nm, GT is 32 ± 0.05°C, EE% is 98.13 ± 0.50%, and Q6 is 33%. Cytotoxicity studies have shown that the safety of the ingredients in the formula is 3.27 times that of the drug. In vitro permeation studies showed a flux enhancement rate of 1.27, indicating an increase in drug permeation from the DLX in-situ cubic gel. In vivo biodistribution studies have shown that IN in-situ cubic gel is much better than IN solution in terms of bioavailability enhancement and brain targeting. Compared with IN solution, the brain bioavailability of IN in-situ cubic gel is increased by 1.96 times. The BTE% and DTP% of the in-situ cubic gel were 137.77% and 10.5%, respectively, indicating effective brain targeting after IN administration. The GMO and Pluronics contained in the cube gel formula have powerful solubilization and penetration enhancement effects, which can improve the encapsulation of DLX in the cube and be directly absorbed into the brain through the nasal cavity. Based on these studies, IN DLX in-situ cubic gel can be regarded as an innovative nano-carrier delivery system for brain targeting of DLX to maximize its effect.

DLX, duloxetine; BCS, biopharmaceutical classification system; CMC, critical micelle concentration; PEO, polyethylene oxide; PPO, polypropylene; CCD, central composite design; GMO, glycerol monooleate; GTP Glycerol tripalmitate; PF127, pluronic F127; PF68, pluronic F68; PS, particle size; PDI, polydispersity index; ZP, zeta potential; GT, gelation temperature; EE%, encapsulation efficiency; Q6 , The percentage released after 6 hours; SD, standard deviation; PBS, phosphate buffered saline; TEM, transmission electron microscope; DSC, differential scanning calorimetry; FTIR, Fourier transform infrared spectroscopy; XRPD, X-ray powder diffraction ; J24, 24-hour drug flux; ER, enhancement rate; IN, intranasal; IV, intravenous injection; IS, internal standard; LC-MS/MS, liquid chromatography tandem mass spectrometry; Cmax, maximum concentration; tmax, peak time ; Kel, elimination rate constant; t1/2, elimination half-life; MRT, average residence time; AUC0-72, the area under the curve from zero to the last time; AUC0-inf., the area under the curve from zero to infinity; BTE%, Brain targeting efficiency; DTP%, the percentage of drug delivery.

The author would like to thank Mr. Dellvin Roshon Williams, an English lecturer in the Department of General Education, Gulf Medical University, UAE, for proofreading and editing the manuscript.

The authors report no conflicts of interest in this work.

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