Polymeric nanoencapsulation of zaleplon into PLGA nanoparticles for enhanced pharmacokinetics and pharmacological activity

Abstract Zaleplon (ZP) is a sedative and hypnotic drug used for the treatment of insomnia. Despite its potent anticonvulsant activity, ZP is not commonly used for the treatment of convulsion since ZP is characterized by its low oral bioavailability as a result of poor solubility and extensive liver metabolism. The following study aimed to formulate specifically controlled release nano‐vehicles for oral and parenteral delivery of ZP to enhance its oral bioavailability and biological activity. A modified single emulsification–solvent evaporation method of sonication force was adopted to optimize the inclusion of ZP into biodegradable nanoparticles (NPs) using poly (dl‐lactic‐co‐glycolic acid) (PLGA). The impacts of various formulation variables on the physicochemical characteristics of the ZP‐PLGA‐NPs and drug release profiles were investigated. Pharmacokinetics and pharmacological activity of ZP‐PLGA‐NPs were studied using experimental animals and were compared with generic ZP tablets. Assessment of gamma‐aminobutyric acid (GABA) level in plasma after oral administration was conducted using enzyme‐linked immunosorbent assay. The maximal electroshock‐induced seizures model evaluated anticonvulsant activity after the parenteral administration of ZP‐loaded NPs. The prepared ZP‐PLGA NPs were negatively charged spherical particles with an average size of 120–300 nm. Optimized ZP‐PLGA NPs showed higher plasma GABA levels, longer sedative, hypnotic effects, and a 3.42‐fold augmentation in oral drug bioavailability in comparison to ZP‐marketed products. Moreover, parenteral administration of ZP‐NPs showed higher anticonvulsant activity compared to free drug. Oral administration of ZP‐PLGA NPs achieved a significant improvement in the drug bioavailability, and parenteral administration showed a pronounced anticonvulsant activity.


| INTRODUCTION
Zaleplon (ZP) is commonly used as a sedative and hypnotic drug with potent anticonvulsant activity. The pharmacological effect of ZP is correlated to its agonist effect on the gamma-aminobutyric acid-A (GABA-A) (type 1) receptor, specifically, benzodiazepine binding sites (Dooley & Plosker, 2000). Based on the biopharmaceutical classification system, ZP is categorized as a Class II drug with poor aqueous solubility and high intestinal permeability. It exhibits a relatively low oral bioavailability of about 30% (Drover, 2004). The low oral bioavailability is a result of combined poor dissolution and extensive first-pass metabolism (Waghmare, Pore, & Kuchekar, 2008). Zaleplon is marketed in two dosage forms: tablets and capsules. However, the oral route of ZP faces numerous obstacles that hinder its oral delivery. The poor aqueous solubility slows down its dissolution with subsequent augmentation of its hepatic metabolism (Dudhipala, 2016). This leads to a disturbance in drug pharmacokinetic represented by delayed onset of action, short elimination T 1/2 (1 h), and short duration of action. Therefore, ZP fails to keep its pharmacological activity reasonable, resulting in early morning awakening (Farag, El Malak, & Yehia, 2018). Increasing the dose of ZP to overcome low oral bioavailability is not recommended because it is related to typically short-lived hallucinations (Farag et al., 2018).
ZP is not among the most often prescribed sedative and hypnotic because it has a quick onset of action and very short elimination half-life of approximately 1 h (Terzano, Rossi, Palomba, Smerieri, & Parrino, 2003). The drug exerts a better action on sleep induction rather than sleep maintenance due to its short half-life and quick onset of action. Previous studies showed that zaleplon has a nonsignificant effect on the total sleep time and the number of awakenings (Sateia, Buysse, Krystal, Neubauer, & Heald, 2017).
Nanotechnology has been revolutionized the field of drug delivery and targeting regarding its supernatural qualities such as small particle size (PS), high exposed surface area, improved physical and chemical stability, and the high biocompatibility of its ingredients (Baker, 2006). Moreover, nanotechnology can optimize the solubility, dissolution, permeability, and bioavailability of several drugs regardless of their physical properties (Card & Magnuson, 2011).
PNPs are colloidal solid particles consisting of natural or synthetic biocompatible polymers formulated into nanoscale particles.
PNPs enjoy great popularity among previously discussed nanosystems for many reasons. First of all, its ease of formulation involves only three simple steps. Second, the simplicity of adjusting and controlling its physicochemical characteristics during formulation.
PNP drug delivery systems can improve the oral bioavailability of poorly soluble drugs, sustain its biological activity, and improve drug stability (Haggag et al., 2020a). Modulation of polymer physicochemical characteristics enabled us to achieve optimal therapeutic efficacy by controlling the optimum release of a therapeutic agent for the required duration needed for attaining the desired therapeutic level in target tissues (Khan et al., 2018).
Moreover, polymeric NP systems may be able to overcome the limitations of nanoemulsions and solid lipid nanoparticles. The major drawbacks of nanoemulsion include low shelf-life stability due to thermodynamics and Ostwald ripening and difficulty in preparation and scale-up production. Formulation of nanoemulsions needs high concentrations of surfactant and cosurfactant necessary for improving the nanoemulsion stability (Patel, Patel, & Thakore, 2018;Yukuyama, Kato, Lobenberg, & Bou-Chacra, 2017). However, solid lipid nanoparticles showed some other disadvantages such as the propensity of lipid oxidation and transformation, incompatibility with various active agents, and a limited drug loading efficiency (Deshpande et al., 2017;Ghasemiyeh & Mohammadi-Samani, 2018).
In contrast, polymeric nanocarriers are more stable in vivo, with high drug loading capacities, as well as controlled or triggered release of drugs (Kamaly et al., 2012). According to these unique properties, polymeric nanomaterials are well positioned in our study to provide a novel solution for ZP oral and parenteral delivery.
Oral delivery is a standard route for drug administration due to its favorable advantages of high patient compliance due to selfadministration ease. However, some physiological barriers control drug bioavailability and therapeutic activity (Bakhru, Furtado, Morello, & Mathiowitz, 2013). The formulation of PNPs is a promising approach to handle these physiological obstacles and enhance the gastrointestinal absorption of drugs with limited aqueous solubility (Jung et al., 2000). The impact of PNPs' size, shape, and surface chemistry largely affects systemic drug delivery after oral administration (Malhaire, Gimel, Roger, Benoît, & Lagarce, 2016).
Antiepileptic drugs can be administrated by different routes. The oral route is the classic route for chronic treatment of epilepsy, but it is challenging to be considered for the treatment of an epileptic attack. Parental, rectal, buccal, and intranasal routes represent the most common alternatives to oral administration, but each route has its advantages and limitations. Parenteral administration is the optimum solution for acute treatment due to the rapid onset of action and successful drug delivery (Musumeci, Bonaccorso, & Puglisi, 2019).
Among numerous existing polymers, poly (dl-lactic-co-glycolic acid) (PLGA) has acquired the interest of several researchers due to its biodegradability, biocompatibility, sustained release profile, and HAGGAG ET AL.
-13 maximum safety issues. PLGA is Food and Drug Administration approved for many formulations for the control of prostate and breast cancers (Crawford & Phillips, 2011;Kamaly et al., 2012). The current study is the first to adopt the formulation of PLGA NPs encapsulating ZP to investigate the bioavailability and pharmacological activity of ZP-PLGA NPs in vivo following oral or parenteral administration.
The following study aimed to inspect the role of PNPs in enhancing the oral administration of ZP as a sedative and hypnotic drug. Besides, improving the anticonvulsant activity after parenteral administration. The effect of various formulation variables, such as the polymer amount, stabilizer concentration, and sonication time, on the characteristics of the nanoparticles, release profiles, in vivo pharmacokinetic behavior, and in vivo biological activity of encapsulated ZP were investigated in this study.

| Fabrication of ZP-PLGA polymeric nanoparticles
ZP-PLGA PNPs were formulated via a simple emulsification-solvent evaporation technique (Khan et al., 2018). To sum up, ZP and PLGA were dissolved in a common organic solvent (DCM) that can dissolve both completely. The selected organic solvent is characterized by its low boiling point, high volatility, high dissolving power, and water immiscibility. The aqueous phase compromised PVA as a stabilizer.
The emulsification process was conducted via an ultrasonic homogenizer with a 3.2-mm probe (Cole-Parmer) to form o/w nanoemulsion. ZP-PLGA PNPs were formed after the evaporation of DCM overnight using magnetic stirring. Finally, NPs were separated by ultra-centrifugation at 30,000g for 30 min with a cooling centrifuge (Sigma Laborzentrifugen GmbH.), followed by three times of washing with ultrapure water and 2% w/v sucrose solution and lyophilized (Labconco). The final product of ZP-PLGA NPs was kept in a desiccator at room temperature. The formulation parameters and identifier codes are listed in Table 1.

| Particle size (PS) and polydispersity index (PDI)
PS and polydispersity index (PDI) were estimated with the aid of the dynamic light scattering principle (Malvern Zetasizer 5000). Briefly, 1 µl of nanosuspension was diluted at 1:10 ratio with Milli-Q ® water after vortexing and sonication. Measurements were recorded as triplicates.
2.3.2 | Zeta potential ZP was recorded using the same equipment of PS and PDI (Malvern Zetasizer 5000). Electrophoretic mobility was used to determine the surface charges of ZP-PLGA NPs. Measurements were represented as triplicates.

| Entrapment efficiency
The entrapment efficiency (%E.E) of ZP was evaluated by an indirect measurement method. The supernatant containing the nonentrapped drug was collected after centrifugation of NPs and used to quantify the free drug using the reversed-phase high-pressure liquid chromatography (HPLC) method (Metwally, Abdelkawy, & Abdelwahab, 2007). In detail, the HPLC system consisted of an autosampler (Waters ® 717), a controller (Waters ® 600), and a tunable absorbance UV detector (Waters ® 486). The mobile phase composed of acetonitrile and water (35:65%) and pumped at a rate of 1 ml/min. The amount of ZP was monitored spectrophotometrically at 232 nm. %E.E was measured by excluding the amount of free ZP from the total amount of ZP and the results were evaluated as triplicates:

| In vitro release study
The release ZP from ZP-PLGA NPs was conducted via the dialysis process Haggag, Ibrahim, & Hafiz, 2020c). Four milliliters of NP suspension was put into a 5-cm dialysis sac (spectra-por, cut-off 12-14 KDa). The dialysis membrane containing the NPs was immersed into 50 ml of PBS release media (pH ¼ 7.4). The medium was stirred at 100 rpm and kept warm at 37 � 2°C using a magnetic stirrer. After each time interval, a 1 ml withdrawn sample was collected and refilled with 1 ml of fresh PBS. The analysis of ZP concentration was performed using the previously discussed HPLC method.

| Sedative and hypnotic action
The sedative and hypnotic effect of ZP was interestingly investigated through the measurement of the effect of ZP and ZP-PLGA NPs on sedation and hypnosis of rabbits (Bellini, Banzato, Contiero, & Zotti, 2014 The MES study was conducted 10 h following intraperitoneal injection of drug treatments. Seizure severity was scored and recorded. The evaluation was based on the extent of tonic-clonic seizure and the extent of tonic extension spread. The scoring system was reported (Wang et al., 2016). The length of tonic extension and occurrence of tonic seizures were also examined as measures of seizure severity (Wang et al., 2016).

| RESULTS AND DISCUSSION
The current study was conducted to investigate the impact of different formulation variables on the various characteristics of the ZP nanoparticles, such as nanoparticles size, surface morphology, and entrapment efficiency. In vitro release to optimize the fabrication of ZP-PLGA NPs having an appropriate size and high drug loading for controlled pharmacological action was investigated.

| Influence of polymer concentration
The w/v to 5% and 10% w/v (Figure 1a). The possible reported explanation of this finding could be due to the higher viscosity of the organic phase.
There is an integral correlation between PS and viscosity of both phases (aqueous and organic phases). Provided that the shear stress is a constant, an increase in the viscosity causes a considerable increase in the resistance to the exposed shear stress. Consequently, the probability of coalescence between nanoparticles during formulation is increased. The balance between agitation shear force and droplet cohesion during the processing of nanoparticles controls the size of formed emulsion droplets (Fude et al., 2005;Haggag et al., 2018a).
Concerning zeta potential, increasing the concentration of polymer to 10% w/v is followed by a significant (p ˂ 0.01) increase in zeta potential, which might be discussed by the intense polymer abundance on nanoparticles' surface following the increase in polymer concentration (Khan et al., 2018). The change in polymer concentration from 2.5% w/v to 5% w/v did not record a significant increase (p ˃ 0.05) in nanoparticles' surface charge in case of (F1, F2) ( Figure 1b).
Drug encapsulation efficiency was significantly (p ˂ 0.05) enhanced by changing the concentration of polymer from 2.5% w/v (F1) to 5% w/ v (F2) and 10% (F3) (Figure 1c). Improvement of ZP entrapment efficiency could be also attributed to the higher viscosity of the oily phase with subsequent enlargement of emulsion droplet size that created a more stable microenvironment to prevent drug escape from the organic phase to the exterior aqueous media. Another possible explanation is the rapid solidification of polymer following the increase in the polymer concentration that restricts drug diffusion from the inner oily phase (Haggag et al., 2016;Yang, Chung, Bai, & Chan, 2000).
The effect of different concentrations of PLGA on the ZP release was shown in (Figure 1d). The early burst effect was interestingly affected by changing PLGA concentrations. A significant (p ˂ 0.05) decline in initial burst release from 24% (F1) to 36% (F3) was detected.
This may be clarified by the fast solidification process that followed due to the high polymer concentration; the polymer matrix will become condensed, resulting in smaller pores and a further tortuous assembly because of the chain entanglement, which obstructs drug transmission to release media (Yang, Chung, & Ng, 2001).

| Influence of the PVA concentration of the aqueous phase
PVA is a frequently used stabilizer in the fabrication of biodegradable PNPs. PVA concentration in the aqueous phase predominately con- with a residual PVA which covered the particle's charge and change the shear plane outwards from the particle surface. 0.5% PVA concentration has the least shielding effect and, consequently, more carboxyl groups are existing for ionization; therefore, the higher zeta potential was obtained . Moreover, a significant (p ˂ 0.05) increase in ZP encapsulation and drug loading was detected after increasing the concentration of PVA in the outer water phase from 0.5% to 1.5% w/v in the case of PLGA nanoparticles (Figure 2c).
This effect might be explained by two hypotheses. First, the increased viscosity of the aqueous phase minimizes the drug transport from the organic phase to the outer aqueous phase. Secondly, the higher PVA concentration resulted in a higher amount of PVA at the interface, which serves as a barrier between the organic aqueous phase that contributed to higher resistance against drug transmission out of the organic phase leading to higher drug loading (Haggad et al., 2016;Khan et al., 2017). However, increasing the concentration of PVA from 0.5% w/v to 1% w/v give rise to nonsignificant (p ˃ 0.05) change in encapsulation efficiency. This might be attributed to lower viscosity of 0.5% and 1% w/v PVA solutions in contrast to 1.5% PVA concentration (Sahoo et al., 2002).
Release behavior of ZP-PLGA NPs formulated with 0.5%, 1%, and 1.5% w/v of PVA were demonstrated in (Figure 2d). The release pattern of (F3) formulated with 0.5% w/v PVA showed a significant (p ˂ 0.05) lower initial burst effect concerning (F4 and F5) prepared with 1% and 1.5% w/v PVA. As long as the burst release was linked to the diffusion of the surface-attached drug, a higher PVA concentration of 1.5% resulted in smaller nanoparticles with higher surface area readily exposed to the media of release which facilitate ZP diffusion and release (Fude et al., 2005;Haggag et al., 2016). sonication times are presented in Figure 3. ZP-loaded PLGA nanoparticles (F7) prepared by using the longest sonication time of 3 min were significantly (p ˂ 0.05) smaller in size than the nanoparticles (F5 and F6) prepared by using 1 and 2 min sonication time, respectively.

| Influence of sonication time
Increasing the sonication time produced a decrease in the PS of F7 (Figure 3a). These findings might be attributed to the higher shear stress used, which would create a suitable condition to prevent coalescence of emulsion droplets resulting in reduced emulsion droplets, which in turn led to smaller nanoparticles (Haggag et al., 2018). Increasing the sonication time from 1 min to 3 min did not count for a significant (p ˃ 0.05) change in nanoparticles' zeta potential ( Figure 3b). However, a sharp (p ˂ 0.05) decrease in drug encapsulation efficiency was observed in F6 and F7 compared to F5 ( Figure 3c). Increasing sonication time resulted in lower drug entrapment. This might be explained by the effect of high shear stress on polymer behavior with disruption of polymeric inner structure and PVA interfacial layer, which facilitates drug diffusion to the aqueous layer (Blum & Saltzman, 2008;Haggag et al., 2018a).
The release profile of (F5, F6, and F7) showed that the drug burst release was faster and efficiently higher from F6 and F7 which released almost 43% and 58% of ZP releases within the initial 24 h on the contrary to 38% of drug released from the F5, respectively ( Figure 3d). The burst release effect is linked to the proportion of the drug that attached the nanoparticle surface (Essa, Rabanel, & Hildgen, 2010). A higher amount of drug was attached to F6 and F7 nanoparticle surfaces compared to F5 because of their smaller PS and higher surface areas. Increasing sonication time leads to the formation of a large number of pores inside the polymeric matrix and, consequently, the drug can quickly diffuse through these pores to the release medium (Bilati, Allemann, & Doelker, 2003).
Screening the previous results, we concluded that F5 showed the highest encapsulation efficiency with a small nanoparticle size of approximately 200 nm, a moderate zeta potential of À 17.5 mV, the highest entrapment efficiency of approximately 96%, and a relatively low burst release of 38%. F5 was used as the optimized formula for further characterization.

| Scanning electron microscopy
The SEM image of F5 was represented in Figure 4. ZP-PLGA NPs had a smooth sphere-shaped appearance with very low PDI. Size measurements by SEM and dynamic light scattering are highly correlated.

F I G U R E 4
Scanning electron microscope images of zaleplonpoly (dl-lactic-co-glycolic acid) nanoparticles (F5) after preparation 3.5 | In vivo study

| Pharmacokinetic study
Zaleplon, being a class II drug, has a poor oral bioavailability as a result of limited water solubility and extensive hepatic metabolism.
The mean plasma concentration versus time profiles of ZP after oral intake of ZP free suspension, ZP marketed tablet, and ZP-PLGA NPs are demonstrated in Figure 5. The measured pharmacokinetic parameters are presented in Table 2. The pharmacokinetic study outcomes clarified that oral delivery of ZP-PLGA NPs (F5) can efficiently alter its pharmacokinetic profile by increasing its bioavailability as compared to the marketed oral tablet and free ZP suspension.  (Du et al., 2018). The formulation of this drug as a nanosystem augmented its solubility and permeability (Xie et al., 2011). In addition, PLGA NPs can reach systemic circulation through gut-associated lymphatic transport, and thus minimizes the hepatic metabolism of the drug (Ahmad et al., 2015). The greater the lipophilicity of nanosystems, the higher the extent of lymphatic transport. These hypotheses, individual and/or in combination, could have donated to the optimization of the bioavailability of ZP from PLGA NPs (Dahan & Hoffman, 2008).

| Assessment of plasma GABA level
The pharmacological activity of ZP depends upon its mechanism of action on (GABA-A) receptors in the brain which increased the GABA concentration (Sanger, 2004). Herein, the plasma GABA level of different animal groups receiving different ZP formulations was measured using a specific rabbit ELISA kit, and the results were 3.5.3 | Assessment of sedative and hypnotic effect ZP is used for the treatment of insomnia due to its agonist effect on the GABA receptor as it indorses sleeping by enhancing the effect of GABA as an inhibitory neurotransmitter (Sanger, 2004

| Evaluation of anticonvulsant activity
The MES model is used for generalized tonic-clonic seizures. This epilepsy model was used to show the efficacy of antiepileptic agents against partial and generalized seizure types. The MES screening tool is useful because it can provide a quick prediction of the anticonvulsant activity of tested drugs with minimal investment and experience (MareŠ & KubovÁ, 2006). In vivo results were represented in Figure 8. A high seizure score was observed for animals treated with free ZP, which was nonsignificant from the control group (p ˃ 0.05) ( Figure 8a). A significant decrease in the stage of seizure was observed in the case of animals that received ZP-PLGA NPs (p ˂ 0.001). The incidence of tonic convulsion was represented for each group (Figure 8b). The number of rats showed tonic seizures/ total rats used and % incidence was calculated for each group of rats.
It is clear that ZP-PLGA NPs exhibit a significant decline (p ˂ 0.001) in the number of convulsed rats with subsequent improvement in % incidence compared to ZP free suspension. Moreover, a marked decrease in the duration of tonic seizures (p ˂ 0.001) was observed for animals treated with drug-loaded NPs compared to control and animal treated with free ZP (Figure 8c). This augmented antiepileptic effect of ZP-PLGA NPs may contribute to the prolonged systemic circulation and sustained drug release from ZP-PLGA NPs, which sustains its pharmacological effect. On the contrary, ZP free suspension is rapidly eliminated from the blood due to its very short elimination half-life.

| CONCLUSIONS
The novelty of this study is the usage of biocompatible PLGA PNPs for oral and parenteral administration of ZP, and in vivo evaluation of its pharmacokinetic behavior and pharmacological activity which is being documented for the first time. Optimizing the physicochemical properties of ZP-PLGA NPs can be achieved through