Materials

Aripiprazole was purchased from Hetero Labs Limited. Chitosan with a molecular weight of 100–300 KDa was obtained from ACROS ORGANICS, USA (Degree of deacetylation ≥ 75%). Tripolyphosphate (TPP) and sodium dodecyl sulfate (SDS) were purchased from ADVENT (Navi-Mumbai, India). Acetic acid, Tween 80, sodium dihydrogen phosphate, sodium monohydrogen phosphate, and sodium hydroxide were purchased by Multi-APEX for Pharmaceutical Industries (Cairo, Egypt). Acetonitrile and methanol (HPLC grade) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA. Dialysis tubing cellulose membrane (molecular weight cut off 12,000–14,000 Dalton) was purchased from Lubrizol Advanced Materials Europe BVBA (Chaussee de Wavre, Brussels, Belgium). Deionized water was obtained from Cairo University laboratories. Other substances used were of chromatographic grade.

MethodsExperimental design

During the preliminary study, many parameters (SDS%, pH values, and Cs: TPP ratio) were selected to investigate their effect on the formulation characteristics (particle size, zeta potential, and entrapment efficacy). Response surface methodology (RSM) involves a list of statistical and experimental techniques used for modelling and analysing the difficulties associated with experimental work [19, 20]. Preparation and optimization of the formulation factors were investigated using a 3-level Box-Behnken design (BBD) expert software (Design-Expert R software version 12, State-Ease, Inc., Minneapolis, MN, USA). Fifteen formulae were prepared based on Design-Expert software using the previously mentioned parameters at three levels for each of them, i.e., low (-1), medium (0), and high (+ 1). The three levels of the three parameters were as follows: SDS concentration (0.00, 0.05, 0.1%), pH(4.5, 5.5, 6.5), and Cs: TPP ratio (2:1, 4:1, 6:1) as shown in Table 1. The impact of independent parameters, namely SDS concentration (X1), pH value (X2), and Cs: TPP ratio (X3), was investigated on the dependent parameters: particle size PS (Y1), zeta potential ZP (Y2), and entrapment efficacy EE% (Y3) as mentioned in Table 2.

Preparation of Ari-loaded Cs-NPs

Ari-Cs-NPs were fabricated using the ionic gelation technique previously described by Murat and Schroën, with slight modifications [21, 22]. Chitosan was dissolved in 1% (v/v) aqueous acetic acid to obtain solutions with different concentrations (10, 6.66, and 3.33 mg/mL), followed by continuous stirring for 20 min to ensure complete dissolution. The pH of the chitosan solution was then adjusted to 4.5, 5.5, or 6.5 using 1 N NaOH (pH/Temperature Bench Mi 150, UK). For nanoparticle preparation, the amount of sodium tripolyphosphate (TPP) was fixed at 10 mg, while the amount of chitosan was varied according to the desired Cs: TPP ratio. For example, at a 2:1 ratio, 20 mg of chitosan was used with 10 mg TPP. The final volume of each formulation was adjusted to 10 mL. The TPP solution was prepared by dissolving 10 mg of TPP in deionized water at a concentration of 2.5 mg/mL, yielding 4 mL of cross-linking solution. Precisely weighed amounts of Ari and different concentrations of SDS (0, 5, or 10%) [22] were dissolved using 0.05% Tween 80 and 0.25 mL of acetic acid. Tween 80 was incorporated to enhance the dispersion of the poorly water-soluble Ari, whereas SDS was included as a solubilizing agent to improve drug incorporation into the chitosan matrix. The drug solution was then added to the chitosan solution under continuous stirring for 10 min on a magnetic stirrer at 1000 rpm (Hot plate stirrer, DAIHAN Scientific Co., Ltd., Korea). Afterwards, the TPP solution was added dropwise to the Ari-Cs solution at a rate of 0.5 mL/min using a syringe, followed by stirring for 1 h at 1000 rpm at room temperature. The resulting nanosuspension was sonicated using a probe sonicator (Sonics Vibra Cell, 130 W, 20 kHz, Sonics & Materials Inc., Newtown, CT) at 80% amplitude for 5 min.

Table 1 Shows the formulation’s variables and measured responses for BBDTable 2 Compositions of the 15 Cs-NPs formulations of BBD and their observed responsesHigh-performance liquid chromatography analysis technique (HPLC) for Ari quantification

An HPLC method (Shimadzu HPLC, Insertsil TM C18 (4.6 × 250 mm, ODS 3–5 μm) column was elucidated to estimate the free Ari in the samples as prescribed by Tushar and Ram Saini [23]. The mobile phase consisted of a mixture of acetonitrile and phosphate buffer in a 70:30 (v/v) ratio. The pH of the phosphate buffer solution was adjusted to 3.6 using acetic acid. The mobile phase was degassed and filtered using a 0.22 μm filter paper. Ari was detected and gave a sharp peak at 254 nm after approximately 2.7 min [24, 25]. The flow rate was 1.0 ml/min with a 20 µl injection volume [26]. The first trial of the sample should be ignored.

Characterization of Ari-Cs-NPsParticle size (PS), polydispersity index (PDI), and zeta potential (ZP)

PS, PDI, and ZP of the freshly prepared Ari-Cs-NPs formulations were measured using Malvern Zetasizer (Malvern Instrument Ltd., UK) at 25 ℃. The samples were diluted 10-fold with deionized water and dispersed in water by gentle mixing. The PS, PDI, and ZP were analyzed in triplicate, and the values are the averages of three measurements ± SD [27].

Determination of Ari entrapment efficiency (EE%)

Ari-Cs-NPs were separated from the dispersion by a cooling centrifuge (HERMLE Labortechnik GmbH, Germany) at 12,000 rpm (≈ 13,680 ×g) for 2 h at 4 °C. After centrifugation, the transparent supernatant solutions were filtered by a 0.22 μm syringe filter and estimated for free Ari using the HPLC analysis technique as previously mentioned. The EE% was estimated according to the following equation [28, 29].

$$EE\%=\frac\text\text\text\text\text\text\,\text\text\text\text\text\text\,\text\text\:\text\text\text-\text\text\text\text\text\text\text\text\,\text\text\text}\text\text\text\text\text\text\,\text\text\text\text\text\text\,\text\text\:\text\text\text}\times100$$

The EE% values are the mean of three estimated values.

Prediction of the optimized formula of Ari-Cs-NPs

The prediction of the Ari-Cs-NPs optimized formula was done after data analysis using Design-Expert-12®software (Design-Expert R software version 12, State-Ease, Inc., Minneapolis, MN, USA) and applying constraints on all the responses to determine the optimum PS, ZP, PDI, and EE%. After that, the optimized formula of Ari-Cs-NPs was prepared, and the evaluated responses were assessed against the predicted values to validate the model. PS, ZP, and EE% were measured for the optimized formula as mentioned before, along with other characterization techniques.

Transmission electron microscopy (TEM)

Morphological analysis of the optimized Ari-Cs-NPs was observed using TEM [3]. The optimized formula was diluted 10-fold with deionized water; after that, a drop of the diluted formula was applied onto a carbon-coated copper grid and allowed to dry at room temperature for 15 min. No staining agent was used before TEM imaging. The sample was then examined by TEM (GEM 1010, JEOL Ltd, Tokyo, Japan) at 80 KV, with a magnification power of 5 KX [30].

In vitro drug release studies

The in vitro drug release of both the optimized formula of the Ari-Cs-NPs and the free Ari suspension (prepared by dispersing Ari in distilled water at the same drug concentration as the optimized Ari-Cs-NPs) was determined using the dialysis bag method [31]. The dialysis bag (Spectra/Pore dialysis membrane with a 12,000–14,000 molecular weight cut-off) was soaked in PBS pH 5.5 for 24 h at 25 °C before being used [32]. For the in vitro release study, 1 mL of Ari-Cs-NPs and 1 mL of Ari suspension, each containing 1 mg of Ari, were placed in the dialysis bag and tied from both ends. This fixed drug amount was selected to enable a standardized comparison of the release profiles between formulations. Each dialysis bag was placed in 100 mL phosphate buffer saline on a shaker bath at 37 ℃ with constant stirring at 100 rpm. At predefined times, 1 mL of sample was collected and filtered using a 0.22 μm syringe membrane filter paper, and the quantity of Ari released was measured using HPLC analysis at 254 nm as previously mentioned [25]. The samples were taken at 1, 2, 3, 5, 7, 12, and 24 h and immediately replaced with freshly prepared media to maintain a constant release volume [33]. The concentration of the released drug at predetermined time intervals was subsequently analysed employing HPLC, as previously mentioned. The amount released was calculated according to the following equation [32].

$$\text\text\text\text\:\text\text\text\text\text\text\text\text\,=\frac\text\text\text\text\text\,\text\text\text\text\text\text\text\text}\text\text\text\text\text\text\:\text\text\text\text\text\text}\times100$$

In vitro cytotoxicity evaluation of the optimized Ari-Cs-NPs

The cytotoxicity of the optimized Ari-Cs-NPs formulation was evaluated using the MTT assay on HFB4 cells (human normal melanocyte cell line) [34]. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine, HEPES buffer, and 50 µg/mL gentamicin. The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂ and routinely subcultured twice weekly.

For the cytotoxicity study, HFB4 cells were seeded into 96-well plates at a density of 1 × 10⁴ cells/well in 100 µL of culture medium and allowed to attach for 24 h. Subsequently, the cells were exposed to serial concentrations of the optimized Ari-Cs-NPs formulation and incubated for an additional 24 h under standard culture conditions. Untreated cells served as the control group. Each concentration was tested in triplicate.

Following incubation, the culture medium was removed and replaced with 100 µL of fresh RPMI-1640 medium without phenol red. Thereafter, 10 µL of MTT solution (12 mM) was added to each well, and the plates were incubated for 4 h at 37 °C in a 5% CO₂ atmosphere. After incubation, a portion of the medium was removed, and 50 µL of dimethyl sulfoxide (DMSO) was added to dissolve the formed formazan crystals. The plates were then incubated for 10 min with gentle mixing.

The absorbance was measured at 590 nm using a microplate reader (SunRise, TECAN, USA). Cell viability was calculated according to the following equation:

$$\text\text\text\text\:\text\text\text\text\text\text\text\text\text\,\left(\%\right)=(\text}_}/\text\text\text)\times100$$

where ODₜ represents the absorbance of treated cells, and ODc represents the absorbance of untreated control cells. The percentage viability was plotted against the tested concentrations to evaluate the cytotoxicity profile of the optimized formulation.

In vivo animal studies

The study protocol was approved by the Ethics Committee of the Faculty of Pharmacy, Cairo University, with approval no. 2900, and was carried out in compliance with the Guidelines for the Care and Use of Laboratory Animals. Wistar albino rats were used in the study. Animals were housed individually in clean cages and given food and water ad libitum with 20% protein, 10% fat, and 70% carbohydrate. The rats were maintained on a 12 h light/dark cycle at a temperature of (20 °C ± 5 ℃) with a relative humidity of (50 ± 10%). They were carefully monitored for any abnormal behaviors during the experiment.

Pharmacokinetic study

Wistar albino rats weighing between 140 and 160 g were divided into four groups (parallel group design). All formulations were administered at an equivalent dose of Ari 20 mg/kg [33, 35]. For IN-administered formulations, the dose was given in each nostril with a micropipette attached to LDPE tubing [32]. Oral solution was administered using a needleless syringe into the mouth of the rats. The intravenous (IV) formulation was administered via the lateral tail vein using a sterile syringe. Group (1) received the Ari solution orally. Group (2) received the Ari solution intranasally. Group (3) was given Ari-Cs-NPs intranasally (the optimized formulation). Group (4) was given IV Ari solution. The rats were kept in a supine position during intranasal administration and even for 3 min after dosing to prevent dose drainage. Ari solution was prepared by dissolving the drug in 0.1% Tween 80, followed by dilution with normal saline (0.9% w/v) to the required concentration. The optimized formulation contained Ari at a concentration of 20 mg/mL. Accordingly, the administered intranasal volume ranged from approximately 140–160 µL per rat, depending on body weight, and was delivered in divided aliquots between both nostrils. Serial blood samples were collected at different time intervals (0.25, 0.5, 1, 2, 3, 4, 6, 8, 24 h) in heparinized tubes and centrifuged at 5000 rpm for 10 min to separate the plasma. To separate the whole brain, at each time interval, 3 rats were euthanized by cervical surgery. The brain tissues were isolated and washed thrice with ice-cold normal saline [36] and then blotted with filter paper to remove any liquids. All brain homogenate and plasma samples were stored at −80 °C until LC-MS/MS analysis [37].

Sample preparation

Before analysis, brain tissues were homogenized in deionized water to obtain the homogenate dispersion using a probe sonicator (Heidolph, Germany). Validated calibration standards of Ari in plasma and brain samples were prepared at a concentration range of (0.5, 2, 5, 20, 50, 500, 70, 90, 100) ng/mL, prepared as follows: 20 µL of IS (Ari d8 as internal standard) and Ari working standard solutions were spiked to 180 µL plasma blank or brain tissues vortexed for 30 s. Intra-batch accuracy and precision were determined using low, medium, and high QC samples. Quality control samples of LLOQ (0.5 ng/mL), Low-QC (1.5 ng/mL), Medium QC-A (10 ng/mL), Medium QC-B (30 ng/mL), and High-QC (80 ng/mL). 20 µL of 100 ng/mL IS in the aliquot of 0.2 mL Plasma or homogenized brain tissue was spiked, vortexed for 30 s, and protein precipitation was carried out by adding 1 mL methanol; samples were vortexed for 4 min, centrifuged at 5000 rpm for 5 min, and then 10 µL was injected into the LC-MS/MS system.

LC/MS/MS analysis

An accurate, sensitive, validated LC/MS/MS analysis technique was utilized for drug analysis in plasma and brain samples [38]. A liquid chromatographic system (Shimadzu, Tokyo, Japan) equipped with degassing unit (DGU-20A3), solvent delivery system (LC-20AB) with an auto-sampler injector (SIL-20 AC) was utilized to inject 10 µL of samples with a flow rate of 0.7 mL/min using 10mM ammonium formate pH 3.5: acetonitrile as a mobile phase with a ratio (55:45% v/v) to a C8 column (Kinetex C8 4.6 × 50 mm × 5 µM). The mass spectrometer (AB SCIEX Model, API-4000, Framingham, MA, USA) was equipped with turbo ion spray operated in positive mode and set at 3000 V with ion source gas at 40 psi and 45 psi for gases one and two, respectively. The common parameters between Ari and its IS were set at 40 psi for curtain and 10 psi for collision gases with a medium temperature of 550℃. The nebulizer gas is air (zero grade), whereas nitrogen is used as the auxiliary, curtain, and collision gas. Data were utilized using Analyst software version 1.6 (SCIEX, Framingham, MA, USA).

Pharmacokinetic parameters

The pharmacokinetic parameters of Ari following nasal administration of the optimized formula, oral solution, IV administration, and intranasal solution were determined using non-compartmental analysis. The peak of maximal concentration (Cmax) and its corresponding time (Tmax) were directly obtained from the brain and plasma concentration-time curves. Moreover, the Linear trapezoidal method was used to calculate the area under the curve for all samples from zero to the last point (AUC 0−24) and to infinity (AUC 0− ∞). The measured concentrations were converted to natural logarithms before calculating the elimination rate constant (Kel), mean residence time (MRT), and the elimination half-life (t1/2). The drug distribution into the brain was calculated using drug targeting efficiency (DTE%) using the following equation [40,40,41].

$$\text\text\text=\frac\text\text\; \text\text\text\text\text/\text\text\text\; \text\text\text\text\text)\:\text\text}\text\text\; \text\text\text\text\text/\text\text\text\; \text\text\text\text\text)\:\text\text}\times100$$

Where AUC brain is the area under the brain Ari concentration-time curve from zero to 24 h, and AUC blood is the area under the blood Ari concentration-time curve from zero to 24 h.

The Percentage of drugs directly transported to the brain via the olfactory and trigeminal pathways can be calculated using the following equation [39].

$$\text\text\text=\frac\text-\text\text}\text\text}\times100$$

Where BX = (BIV/PIV) × PIN.

Where BIN and PIN are the areas under the curve from zero to 24 h in brain homogenate (B) and plasma (P), respectively, following IN administration of the optimized formula of Ari-Cs-NPs. On the other hand, BIV and PIV are the areas under the curve from zero to 24 in brain homogenate (B) and plasma (P), respectively, after IV administration.

Additionally, the absolute bioavailability/brain bioavailability (F) of Ari after nasal administration was calculated using the following equation [42, 43].

$$F=\frac\:\text\text\times\text\text\text\:\text\text}\:\text\text\times\text\text\text\:\text\text}$$

Absolute bioavailability: where (D) is the dose of Ari, (AUC IN0–∞) is the area under the concentration-time curve after IN administration in the plasma, and (AUC IV0–∞) is the area under the concentration-time curve after IV administration in the plasma.

Brain bioavailability: where (D) is the dose of Ari, (AUC IN0–∞) is the area under the concentration-time curve after IN administration in the brain, and (AUC IV0–∞) is the area under the concentration-time curve after IV administration in the brain.

Pharmacodynamic and behavioural studies

Induction of psychosis in animal models was performed by using ketamine at a sub-anaesthetic dose (30 mg/kg i.p./day) for five consecutive days [44, 45]. All formulations were administered at an equivalent dose of Ari (20 mg/kg/day) for ten successive days. Wistar albino rats weighing between 140 and 160 g were divided into six groups (parallel group design). Group (1) was a negative control group (normal control) that received 0.9% norml saline. Group (2) was a positive control group of schizophrenia-induced rats by ketamine (untreated group). Group (3) received the void formula (without drug) intranasally. Group (4) received the Ari solution intranasally. Group (5) received the optimized formula of Ari-Cs-NPs intranasally. Group (6) was given the Ari solution orally. Ari solution was prepared as previously mentioned. The optimized Ari-Cs-NPs formulation contained Ari at a concentration of 20 mg/mL. The required dose was administered intranasally in divided aliquots into both nostrils, with the total volume adjusted according to the body weight of each animal. Animals in all groups were subjected to behavioral assessment tests (open field test and forced swim test) to detect the effectiveness of treatments in the ketamine-induced psychosis model. After the evaluation of behavioral parameters, animals were sacrificed, and brain samples were collected, washed, and divided into two equal parts. One part was used to investigate brain biomarkers, while the other part was used for histopathological examinations.

Open-field test

The open-field test was used to investigate the locomotor activity and rats’ exploratory behavior. Briefly, rats were carefully placed on one side of the wooden square (70 × 70 × 35 cm) and kept for 3 min. The total counts of crossing squares, and rearing frequency were measured [44, 46]. The locomotor activity was performed 24 h after the administration of the last dose. The measuring apparatus was cleaned with 70% alcohol after each measurement.

Forced swim test

The forced swim test is considered a definitive tool to determine the negative symptoms in rats introduced in the ketamine model (depression-like behavior). The procedure was divided into two sessions, 24 h apart. The first session is a training part where the rat was placed into a glass chamber with a height of 15 cm containing water (23 ± 2 °C) for 15 min. Afterward, the rat was subjected to a 5-minute testing session. Mobility duration (swimming and climbing), in addition to immobility duration, was measured. During the testing part, the first minute of the measurement was ignored due to the animal’s acclimation. A forced swim test was performed 48 h after the last administration [47, 48].

Brain biomarkers and neurochemical estimations

Dopamine (DA) and γ-aminobutyric acid (GABA) concentrations were measured in rats’ brains by competitive enzyme-linked immunosorbent assay (ELISA) using Rat DA and Rat GABA ELISA kits (CUSABIO, USA) according to the manufacturer’s protocol. Brain samples weighing 300–500 mg were rinsed in ice-cold phosphate buffer saline (PBS) (0.02 mol/L, pH 7.0-7.2) to ensure the complete removal of residual blood. The brain tissues were homogenized in 500 µl of PBS using a homogenizer under ice conditions. The resulting suspension was subjected to ultrasonication for 5 min to facilitate efficient disruption of brain tissue and cellular structures, thereby maximizing the release of intracellular neurotransmitters. After that, the brain homogenates were centrifuged at 5000 rpm (≈ 2,375×g) for 15 min at 4℃ to obtain a clear supernatant for subsequent ELISA analysis. All samples were processed under identical conditions according to the manufacturer’s instructions to ensure consistent neurotransmitter recovery and minimize experimental variability. The results were expressed as ng/mg.

Measurement of dopamine levels (DA)

The rat DA ELISA kit applies the competitive enzyme immunoassay technique utilizing the polyclonal anti-DA antibody and a DA-HRP (avidin-conjugated horseradish peroxidase) conjugate, as mentioned by Badawi et al. [49], . 100 µl of the sample was incubated with 10 µl DA-HRP conjugate in a pre-coated plate for 1 h at 37℃. After the incubation period, the wells were washed and then incubated with 50 µl substrate for the HRP enzyme (TMB). The product of the enzyme-substrate reaction gives a blue complex. Finally, a stop solution is added to stop the reaction and give a yellow solution. The intensity of the color is measured using the spectrophotometer at 450 nm.

Measurement of γ-aminobutyric acid levels (GABA)

The pre-coated antibody is an anti-GABA monoclonal antibody, while the detection of the antibody is a biotinylated polyclonal antibody [50]. 100µL of brain samples and biotinylated antibodies are added into the ELISA plate wells for 60 min at 37℃. Then, Avidin-peroxidase conjugates are added to the wells. TMB substrate is used for coloration after the enzyme conjugate has already been washed out of the wells with PBS. TMB reacts to form a blue mixture from the peroxidase activity and finally turns yellow after adding the stop solution. The intensity of the color can be detected at 450 nm using a spectroscope [50].

Effects of IN Ari-Cs-NPs and other Ari formulations on ketamine‑induced histopathological alteration in rats’ brains

The brains were isolated from rats after the induction of the model and preserved in 10% v/v formalin for 72 h, embedded in paraffin containing hematoxylin and eosin (H&E) staining for pathological examination [51]. 4 μm-thick sagittal brain sections were cut by the rotatory microtome for the demonstration of the hippocampus and cortex regions in all brain samples. All light microscopic examination and data were obtained using CX 41RF, Olympus Corporation, Tokyo, Japan, attached to a full HD microscopic imaging system, Olympus soft imaging, SC100, made in Germany.

Statistical analysis

Each experiment was repeated three times, and all results were expressed as the mean ± standard deviation (M ± SD). To establish a statistically significant difference, one-way analysis of variance (ANOVA) and multiple comparisons (Post Hoc Tests) will be used to compare different groups using the statistical software IBM SPSS STATISTICS Version 26. Pharmacokinetics results were calculated using PKanalix 2024R1, Lixoft SAS, a Simulations Plus company, USA. Observed responses were deemed significant at a p-value < 0.05.