A pure OLZ powder was obtained as a free sample for laboratory use from Hyperchem, China. Soluplus® polymer was purchased from BASF, Germany. Polyvinyl alcohol (PVA) was obtained from Rhom, Pharma, Germany. Polyvinylpyrrolidone (PVP) with different grades, like (PVP-K15), (PVP-K30) were purchased from Provizer Pharma, India. The methanol solvent was from Sigma-Aldrich, Germany. All solvent and chemicals of analytical grade were used.

OLZ nanoparticles were formulated by the nanoprecipitation technique (solvent/antisolvent). The formulation occurs through the utilization of organic solvent miscible with water, which is methanol 3 ml for solubilizing olanzapine powder 10 mg, then the produced organic phase was injected dropwise at a rate of 1 ml/min by a syringe pump into 30 ml of aqueous phase (deionized water with stabilizer) under continuous stirring (600 rpm). Upon dropping, the precipitation of nanoparticles occurs promptly, producing nanosuspension. The remaining organic solvent was evaporated by using a magnetic stirrer 1 hrour at 30 °C (Alwan and Rajab 2021; Al-Mahmood and Abd Alhammid 2022). The composition of (OLZ) nanoparticles is listed in Table 1.

Particle size and polydispersity index (PDI) measurement

The size of particles and PDI were screened using a Malvern zetasizer instrument (Malvern, UK). The size was measured by the zeta sizer through the determination the intensity of scattered light by particles found in the sample with a scattered angle of 90 °C at room temperature. Also, PDI was measured by a zeta sizer, which gives an indication of the distribution and uniformity of particle size within the sample (Hamed and Hussein 2020). A triplicate reading was recorded.

Zeta potential measurement

The zeta potential of formulated nanoparticles was measured by a Malvern zetasizer instrument (Malvern, UK), and its value gives an indication about the stability of nanodispersion (Anarjan et al. 2014; ALedresi et al. 2020).

Entrapment Efficiency (EE %)

The measurement of the entrapment efficiency of olanzapine nanoparticles was made by utilizing the indirect method. By this method the concentration of free olanzapine present in the dispersion medium was measured, and this was done by putting 5 ml of drug nanodispersion in an Amicon® Ultra Centrifugal tube with a molecular cutoff (MWCO) of 10 kDa followed by centrifugation at 3000 rpm for 20 min (Yongjiu et al. 2018). The dilution of unentrapped OLZ found in ultrafiltration and measurement was made by a UV-visible spectrophotometer at 270 nm (Ruby and Pandey 2016), by using the following equation:

$\text{E E}\%=\frac{\text{WT}-\text{WF}}{\text{WT}}\times 100$

Where, EE% is entrapment efficiency, WT is the total weight of drug used, WF is weight of free olanzapine that is measured in the supernatant layer after ultrafiltration. The measurement was made in triplicate and the values expressed as mean±SD.

In vitro release profile of nanoparticles

The release study of OLZ as nanodispersion and as a pure drug was carried out by putting an adequate volume 9 ml of OLZ nanodispersion containing 3 mg of drug and 3 mg of pure OLZ powder drug in a dialysis bag 8000–14000 Da (Hi Media Lab Pvt. Ltd India) (Dalvi and Dave 2009). Then the sealed dialysis bag was immersed in 500 ml phosphate buffer pH 7.4 contained 0.5% tween 20. The process was performed by the utilizing dissolution apparatus USP-II (paddle) at 37 °C±0.5 with a speed of 50 rpm. Sample volume 5 ml were taken at time intervals of (5, 10, 15, 20, 30, 40, 50, 60, 70, 80 and 90 min), and each withdrawn volume was replenished by buffer to maintain sink condition. Then filtration by membrane (0.45 µm) was performed, and the concentration was measured by a UV-visible spectrophotometer at 252 nm (Joseph et al. 2015). A triplicate measurement was done.

Surface morphology of nanoparticles

The surface morphology of nanoparticles was determined by using a field emission scanning electron microscope. (FESEM) (HITACHI S–4160, Japan).

Differential scanning calorimetry (DSC)

DSC is a thermal technique performed by putting an adequate amount 5 mg of pure OLZ powder, physical mixture of drug with polymer, and optimized lyophilized nanoparticles in the aluminum pan of (DSC-60 Shimadzu, Japan) with heating at a rate of 10 °C/min at 50 to 250 °C, and nitrogen flow of 40 ml/min (Polla et al. 2005; Antunes 2010).

MNs Fabrication

The dissolving MNs mold contains 225 conical needles, which are arranged in (an array size of 15×15) with height of 500 μm, a base diameter 200 μm, and a needle pitch of 1500 μm that is located in approximately 4 cm² area. The mold was purchased from Micropoint Technologies Pte Ltd. Singapore.

Preparation of the MNs matrix

The polymers utilized to prepare the dissolving microneedles matrix are PVP-K30 and PVP; hence, the polymeric solution was formulated by dissolving 2 gm either of each polymer or a combination of two polymers with different ratios in 20 ml of distilled water, then glycerin 5% (w/w) as plasticizer was added to the water and heated at 50 °C for 2 hours. The produced polymeric solution was placed in a sealed glass container overnight to obtain a solution free from bubbles to be used in fabrication of dissolving MNs (Noor and Ghareeb 2021). Table 2 explains the composition of MNs formulas.

Drug loading in MNs mold

Loading of OLZ nanosuspension (1 mg/3 ml) into mold cavities using sonication for 1 hour then leaving the mold in a degassed desiccator for 24 hours for drying. After that, the polymeric solution is cast into the mold and sonicated for 30 min, then left the mold in degassed desiccator for 24 hours for drying. The drug will be deposited in the maximum amount at the tip of the MNs (Chu et al. 2010). The fabrication steps of OLZ nanoparticles as dissolving MNs were explained in Fig. 1.

MNs morphology

The morphology of dissolving MNs can be observed by using a digital microscope (Depstech, China) to determine the dimension uniformity of MNs, like length, width, and interspacing of MNs. Also, the morphology of optimized MN was performed by scanning electron microscope SEM (TESCAN, UK).

Drug Content

The drug content in the MNs patches was screened by putting the patch (2.5 cm × 2.5 cm) in 50 ml (25 ml methanol with 25 ml DW) on a magnetic stirrer for 3 hours. Then filtration by filter membrane (0.45 µm) and dilution with methanol, the amount of drug is measured by using UV-visible spectrophotometer at 270 nm (Badshah et al. 2010).

Formulation of simple patch of OLZ-NP

A simple patch of OLZ nanoparticles can be formulated by the same method that is utilized in the preparation of microneedles. That means the first step involves casting the nanodispersion in a petri dish and allowing it to dry; the second step involves casing the polymeric solution and allowing it to dry; and then cutting the simple patch into dimensions similar to those of a microneedle patch.

Ex-vivo permeation of drug

Skin from the abdomen of a male rabbit weighing 1.25 kg±0.14 was taken from the animal house in the college of pharmacy, university of Baghdad, for the purpose of conducting an ex vivo permeation of nanoparticle- loaded dissolving MNs and a simple patch of OLZ-NP. This study was performed using a Franz diffusion cell in which the skin is placed between the donor and receptor compartments, with facing the stratum corneum to the upper side (Al-Hamadani and Al-Edresi 2022). The size of MNs patch is 6.25 cm² containing 1 mg of OLZ, but the effective skin surface area that is available for diffusion of the drug in Franz cell is 3.14 cm², so, the amount of OLZ available for diffusion was 0.5 mg. The receptor compartment is filled with 25 ml of phosphate buffer pH 7.4 with 0.2% (w/w) tween 20 to maintain sink condition. The receptor medium was continuously stirred at 100 rpm at temperature of 37 °C. The dissolving MNs and a simple patch of OLZ-NP containing 1 mg of drug were placed on skin by gentle pressure. Sample 2 ml withdrawn from receptor compartment at time intervals (15, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270 and 300 min), and substituted with fresh medium to maintain sink condition. Samples analysis was made by using UV-visible spectrophotometer at 252 nm. By plotting the cumulative amount of permeated drug versus time interval, the slop of the curve represents steady-state flux (Jss) of drug (Drais and Ahmed 2022; Atiyah Altameemi and Abd-Alhammid 2022).

Pharmacokinetics study

Firstly, blank plasma can be obtained from male rabbits as a negative control for high performance liquid chromatography (HPLC) analysis. Then twelve male rabbits weighing (1.7±0.15 kg) were separated into two groups (n=6 in each group). Group1 includes the application of dissolving MN patch on the dorsal surface of the rabbit after shaving hair and clearing with spirit as in Fig. 2. Group II includes oral administration of the drug by using a conventional tablet of OLZ. The dose of drug for each group, transdermal and oral dose was calculated depending on body surface area by using the following equations:

AED (mg kg–1) = HED (mg kg–1) x Kmratio

Where, AED is animal equivalent dose, HED is human equivalent dose:

${\text{ Km}}_{\text{ratio}}=\frac{{\text{Human}}_{\text{Km}}}{{\text{Animal}}_{\text{Km}}}$

Where K_m is a factor that is obtained from dividing reference body weight (kg) by body surface area (Nair and Jacob 2016), K_m factor is different across animal species and proportionally increases as body weight of species increases, K_m for human can be calculated by dividing reference body weight of human 70 kg over body surface area of human (1.72 m²), so, K_m of human is about 40.69 and K_m of rabbit weighing 1.7±0.115 kg is 11.3 (Jacob et al. 2022). Transdermal (HED) is 0.102 mg /kg, and oral (HED) is 0.142 mg/kg. So, the transdermal (AED) and oral (AED) that have been administered for group1 (MNs) and group II (oral) are 0.622 mg and 0.869 mg, respectively. In both groups I and II, the animals were sedated gently with ether, blood samples were collected from jugular vein by using 1 ml syringe (25 gauge) at time intervals of (0.5, 1.5, 2.5, 4, 6, 12, 24, 48, 72, and 96 hour), so, 0.5 ml of blood sample was withdrawn at each time interval and placed in microcentrifuge tube (contain EDTA) to avoid clotting, after collection of samples, centrifugation at 3000 rpm for 10 minutes and separating the plasma with micropipette, then plasma samples were stored at -20 °C for later analysis.

Analysis of OLZ concentration in plasma

The analysis was performed by utilizing HPLC system S600- Sykam GmbH (Germany) provided with hypersil-BDS C18 column (250–4.6 mm I.D, 5 μm) for separation. Mobile phase composed of mixture of Methanol-Acetonitrile- phosphate buffer of 50 mM at pH (5.5) (20:30:50) (v/v/v) which was run at the flow rate of 1 ml/ min with run time 10 minutes. OLZ wavelength detection was 254 nm (Dusci et al. 2022).

Sample preparation

Extraction of OLZ from samples of rabbit plasma was made by using liquid-liquid extraction. 0.1 ml of plasma placed in a 10 ml borosilicate glass tube, followed by addition 10 μl of internal standard (I.S.) solution, which is (5 μg /ml of fluoxetine) into biological sample, then 5 ml solution of dichloromethane: hexane (20:80) was added and the mixture was blended on vortex mixer for 5 minutes and centrifuged at 3000 rpm for 10 minutes. Supernatant layer of the mixture was placed in 10 ml glass tube. Then mobile phase (100 μl) was added and blended by vortex mixer. 100 μl of resulting sample was injected to HPLC for OLZ analysis (Pervaiz et al. 2015). Pharmacokinetic parameters, which are maximum plasma concentration (C_max), Area under curve (AUC) and the time required to obtain maximum plasma concentration (T_max) were calculated.

Preparation of stock solutions

OLZ and fluoxetine stock solutions were prepared by using methanol at concentration 1 mg/ ml. Working standard solutions were prepared by dilution of stock solutions with methanol. OLZ standard solutions were 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.3 μg/ml, while, fluoxetine standard working solution was 0.01 μg /ml.

Linearity

Preparation of calibration curves was made by addition 10 ml of standard working dilutions of olanzapine and fluoxetine to 0.1 ml drug free rabbit plasma. So, plasma calibration standards with concentrations of 5, 10, 20, 50, 100, 200, 300 ng /ml were obtained. Calculation of standard calibration curves was made by using the ratio of peak area for olanzapine and that of fluoxetine as function of olanzapine plasma concentration.

Recovery

The estimation of recovery was done by dividing the concentration extracted OLZ over concentration of non-extracted and multiplying by 100%. Three concentrations 80, 100 and 150 ng/ml were utilized in recovery method (D’Arrigo et al. 2006).

Precision and accuracy

Intra-day and inter-day precision were expressed as relative standard deviation (RSD%), and accuracy was expressed as percent error (%). The precision was estimated by measuring the concentration of spiked plasma (10 ng/ml) for OLZ three times per day (n=3) as intra- day precision and for three days (n=3×3) as inter-day precision (Olesen and Linnet 1998).

Statistical analysis

The results of three independent experiments were performed and analyzed using Excel 2016. The results are expressed as mean with standard deviation. One-way analysis of variance (ANOVA) was performed as appropriate. The results were considered statistically significant at P<0.05.

The results of measurement of particle size reveal that all formulated OLZ nanoparticles are present in nano-scale size with a range 77.98 to 344 nm, and the results of PDI were in the range 0.152 to 0.404, as shown in Table 3. PDI gives an important idea that concerns with uniformity and distribution of nanoparticles within a sample. So, the interpretation of PDI values revealed that the sample is monodisperse standard with a PDI range of (0.0–0.05), the sample is nearly monodispersing with a PDI range of (0.05–0.08), mid-range polydispersity (0.08–0.7), and the sample is very polydisperse with a PDI value greater than 0.7 (Hussien and Ghareeb 2021). So, the results of PDI below 0.7 that mean all olanzapine nanoparticles havemid-range polydispersity.

The results of the measurement exhibit that all nanoparticle formulations have lower zeta potential values, which are in the range of -3.75 to -19.01 mV as found in Table 3. Low values of zeta potential give an indication about existence of steric stabilization within the formulation, through which the adsorbed stabilizer layer causes a shift for the shear plane far from particle surface, which is considered the place of zeta potential analysis; hence, the value of zeta potential -19.01 mV for nanoparticles (NP-5) is appropriate for stabilization due to the steric effect that is present along with electrostatic stabilization that is produced by zeta potential (Xu et al. 2022).

The results of particle size measurement exhibited that all formulations of olanzapine nanoparticles were in the nanoscale range; this indicates that the polymers utilized in the study don’t have an influence on particle size, but there was a significant relation between the amount of polymer and particle size, as shown in Table 3. This means that when the drug: polymer ratio of nanoparticle formulations is raised; the particle size will be increased. This may occur due to the increased viscosity of the medium, which restricts the movement of particles within the solution and prevents better covering of recently formed nanoparticles. Also, a high amount of polymer results in an increase in the thickness of the coat that covers the nanoparticle and avoids the diffusion between solvent and anti-solvent during the precipitation of nanoparticles (Liu D et al. 2012). Both types of polymers that produce olanzapine nanoparticles have mid-range polydispersity because the PDI values in all formulations were lower than 0.7 (Hussien and Ghareeb 2021; Al-Edresi et al. 2024), as shown in Table 3. The effect of polymer type on zeta potential can be described by the ionization of polymer in the medium, and this depends on the pKa of the polymer. The results in Table 3 indicate that both polymers exhibited little ionization in the medium of nanodispersion and resulted in lower zeta potential values. This mean that pKa of the polymer and the pH of the medium are responsible for zeta potential values, Soluplus® exhibits higher ionization and zeta potential values as compared with PVP-K30 (Barbosa et al. 2019).

The results of the entrapment efficiency of nanoparticles exhibit a relevance between stabilizer (polymer) ratio and EE%, which means there was a significant increase (P<0.05) in EE% when stabilizer amount increased. The interpretation of this idea is that when the stabilizer ratio increases, there is an improvement in polymer attachment to the nanoparticle shell, which results in ameliorating the hydrophilic characteristics of the nanoparticles. Consequently, a lower diffusion of drug into the medium causing a higher EE% (Ismail et al. 2015). Table 3 explains the EE% of formulated nanoparticles with different polymers ratios.

Depending on the characterization studies of the prepared nanoparticles, which are particle size, zeta potential, and entrapment efficiency, three formulations (NP-2, NP-3, and NP-5) were selected for performing an in vitro release. The results reveal that all formulations exhibited higher and more significant drug release (P<0.05) as compared with pure drug. Particle dissolution rate is considered a function of particle surface area, and this is described by Noyes-Whitney equation, which explains that as the particle size decreases, the solubility will improve thereby enhancing the dissolution rate. This interpretation is compatible with the results that gained by Ali and Abd-Alhammid (2019), when they prepared a nanosuspension of atorvastatin calcium using different polymers by nanoprecipitation. The results exhibit the amelioration in the dissolution rate of a pure drug when designed as a nanoparticle, and cumulative release was 44% and 90% for a pure drug and formulated nanoparticle, respectively (Ahmed and Shaimaa 2019). Also the type of polymer has an influence on drug release from nanoparticles, and this is based on polymer-drug interaction. Consequently, the Soluplus® is co-polymer with amphipathic properties that act as a wetting agent and surfactant, which causes a lowering in interfacial tension between the surface of the nanoparticle and the anti-solvent. So, Soluplus® permits surface-water interaction that maintains the lowest size of nanoparticle there by fast dissolution and release of drug as compared with other stabilizers, and this indicates that NP-5 exhibits a higher and more significant release of OLZ (P<0.05) as compared with other formulations of PVP-K30, and pure drug. An in vitro release of active pharmaceutical ingredient (pure drug) exhibits a lesser significant release (P<0.05) as compared with the formulations of nanoparticles for both type of polymers, Soluplus® and PVP-K30, due to poor water solubility of active pharmaceutical ingredient (API) (Yang et al. 2014). Fig. 3 reveals the in vitro release profile of OLZ nanosuspensions.

Depending on the results of characterization studies of OLZ nanoparticles like particle size, entrapment efficiency, PDI and zeta potential, it can be concluded that NP-5 is considered as optimized formula due to good particle size 115.76 nm as in Fig. 4, the high entrapment efficiency 78.4% of optimized formula (NP-5) due to the better attachment of Soluplus® into the nanoparticle shell, and result in promoting the viscosity of stabilizer solution, thereby slowing the diffusion of drug into external phase, and this results in good entrapment efficiency, PDI value 0.24 that exhibits uniform particle size within formula, higher value of zeta potential -19.01 mV as in Fig. 5, higher, and more significant drug release (P<0.05) as compared with other formulations and pure drug, so, NP-5 can be utilized in fabrication of dissolving MNs patches.

The morphology of nanoparticles (NP-5) was screened by using field emission scanning electron microscope (FESEM), and the result revealed that nanoparticles have spherical shape and size approximate to that observed by zeta sizer as in Fig. 6.

The results of DSC analysis detect a sharp endothermic peak for OLZ powder at 198.38 °C, which is identical to the reference reading of the melting point of the drug (196 °C to 198 °C) (Rudrangi et al. 2015; Patil et al. 2023). The DSC of the physical mixture (drug: polymer) exhibits a two endothermic peaks for drug and polymer, and this reveals the compatibility that is present between drug (olanzapine) and polymer (Soluplus®). DSC of lyophilized nanoparticles (NP-5) exhibits an endothermic peak with shifting due to the decrease in the crystallinity of drug (Cho et al. 2020). The DSC thermogram of pure olanzapine, the physical mixture, and OLZ nanoparticle (NP-5) explained in Fig. 7 A–C, respectively.