Enhancing Intranasal Delivery and Bioavailability of Dihydroergotamine Utilizing Chitosan Nanoparticles

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Introduction
Migraine is a chronic headache disorder with a major impact on the lifestyle of patients. It has been found to be the second leading cause of disability in the United States regarding "impaired quality of life, substantial lost productivity, and high-economic costs" [1]. Approximately 17% of women and 6% of men in the United States were reported to sufer from migraines [2]. One study in Jordan found that around 7.7% of adults experience migraines [3]. Migraine patients usually prefer medications with rapid onset and fast pain relief [4][5][6]. Furthermore, many migraines present with nausea, which makes swallowing oral medications difcult, requiring alternative dosage forms like intranasal sprays [7]. Dihydroergotamine (DHE) ((5′α)-9,10-dihydro-12′hydroxy-2′-methyl-5′-(phenylmethyl)-ergotaman-3′,6′,18trione) ( Figure 1) is a chemical derivative of an ergopeptine core alkaloid. It binds to serotonin 5-hydroxytryptamine 1 (5HT 1 ) receptors, making it a highly efective medication for the treatment of migraine [8]. DHE has been found to be helpful with rebound headaches, chronic daily headaches, menstrual migraines, and in patients not responding to triptan treatment [9,10].
Te drug has low oral bioavailability. Oral DHE is extensively metabolized by the frst-pass efect; therefore, it must be given by a nonoral route. Furthermore, it has incomplete and inconsistent drug passage across the gastrointestinal mucosa, making it suboptimal for clinical use [11].
Parenteral DHE requires healthcare personnel to administer, and it is challenging to self-administer. Tis demands the development of innovative strategies to overcome these difculties. Te nasal route is an attractive modality to enhance therapeutic efcacy and to reduce the extent of the frst-pass efect. Furthermore, alternative drug delivery systems have been proposed such as inhalations, microneedles, and injections, which bypass the liver and are more rapidly absorbed [10,[12][13][14][15].
Chitosan is a mucoadhesive polysaccharide derived from partially acetylated chitin [36] that has been often prepared as nanoparticles to improve intranasal delivery of drugs [37][38][39]. It is mucoadhesive and has been shown to enhance drug permeability across membranes [40], thus making it a promising candidate for intranasal drug delivery.
Te marketed nasal DHE spray has low systemic bioavailability (32%) [41] and intersubject diferences in selfadministration, along with reported spillage [42] which can lead to drug loss, disturbed taste, and abdominal pain as it runs out of the upper lip or down the back of the nasopharynx, leading to suboptimal therapeutic efects [42][43][44], and rhinitis, which is a common adverse event [41].
Te aim of our work includes the formulation of DHE in chitosan nanoparticles for intranasal application to enhance systemic absorption. To our knowledge, this is the frst study to investigate DHE intranasal delivery with nanotechnology in an anesthetized rat model.

Preparation of Chitosan Nanoparticles.
Chitosan nanoparticles (CS-NPs) were prepared based on a modifed ionotropic gelation method of chitosan with tripolyphosphate (TPP) polyanions [45]. Chitosan (200 mg, 0.20% w/v) was dissolved in 100 mL of 3% (v/v) acetic acid with the pH adjusted to 4.7 using 10 N NaOH. Ten, TPP aqueous solution (0.7 mL, 0.2% w/v) was added stepwise to 1.5 mL of chitosan solution (0.2% w/v) under magnetic stirring (700 rpm) at room temperature. In order to load chitosan nanoparticles with DHE, DHE was incubated in the initial CS solution for 4 minutes, followed by the addition of the TPP solution as described above.
CS-NPs were loaded with diferent concentrations of DHE: 0.5 mg, 0.99 mg, and 1.6 mg (20%, 30%, and 40% theoretical loading). Additionally, diferent ratios of CS: TPP and loading mechanisms were evaluated. Te resulting best ft with 20% DHE loading was utilized for the in vivo study.
Nanoparticle solutions were centrifuged in centrifuge tubes at 20,000 × g for 20 min at controlled room temperature (CRT), and then the supernatant was discarded. Te resulting nanoparticles were lyophilized until use.

High-Performance Liquid Chromatography (HPLC)
Assay. Analysis of DHE was performed on a Shimadzu reversed phase high-performance Liquid chromatography (RP-HPLC) system (model UV 1601 PC, Kyoto, Japan) equipped with an Agela-Unisol RP-C18 column (4.6 × 150 mm, 5 μm) and Vp 6.14 software. Te mobile phase consisted of water, acetonitrile, triethylamine, and trifuoroacetic acid (70 : 30 : 0.1 : 0.1) (pH 2.5) delivered at a fow rate of 1.5 mL/min at room temperature. For aqueous samples, the UV/VIS detector was set at 280 nm, and plasma samples were analyzed with fuorescence detection at Ex 280 nm and Em 350 nm for enhanced sensitivity.

Morphological Examination.
A scanning electron microscope (SEM) was used to evaluate the morphology and particle size distribution of the DHE-loaded CS-NPs. Freezedried nanoparticles were coated with 2 nm gold at room temperature. Te grid was examined with feld emission gum-scanning electron microscopy (FEG-SEM) (FEI QUANTA FEG 450).
Particle size and zeta-potential of CS-NPs were determined using dynamic light scattering using a Zetasizer (Malvern Instruments, Malvern, UK).

Determination of Entrapment Efciency (EE) and Drug
Loading (DL) Percentages. Te DHE-loaded nanoparticles were separated from the aqueous medium by ultracentrifugation at 20,000 × g for 20 min at CRT (Beckman centrifuge, Fullerton, Canada). Te clear supernatant was diluted in triplicate and analyzed for free nonentrapped DHE by high-performance liquid chromatography-UV detection as described above. Te diference between the amount of DHE used for nanoparticle preparation and the free nonentrapped DHE in the supernatant was considered as the amount of DHE loaded into the nanoparticles.
Te efciency of drug encapsulation efciency (EE) and drug loading (DL) of nanoparticles were calculated according to the following equations: EE% � [(total weight of DHE added − weight of free nonentrapped DHE)/(total weight of DHE added)] × 100.

In Vitro Release of DHE from DHE CS-NPs.
Sink conditions for the drug release studies were assessed using small portions of nanoparticle suspension containing 1 mg of DHE and diluted in 10 mL of purifed water. Samples were then incubated at 37°C and agitated (500 rpm). At predetermined time intervals, 1 mL samples were withdrawn and replaced by fresh phosphate bufer media. Te samples were centrifuged (18,000×g for 20 min) and the released DHE was determined by HPLC/UV.

In Vivo Nasal Bioavailability Studies.
Te nasal absorption of DHE and DHE-loaded nanoparticles containing 20% DHE were studied in vivo using male Sprague-Dawley (SD) rats (Animal House Institute, Jordan University of Science and Technology) weighing 260-350 g (n � 9) [27,28]. Tis research was approved and monitored by the Institutional Animal Care and Use Committee (IACUC) of Jordan University of Science and Technology prior to its initiation and during its execution.
All surgical procedures were performed under anesthesia (intraperitoneal injection of xylazine 2% (0.1 mL) and ketamine 10% (0.1 mL)). Te rat's trachea was cut and separated from the gastrointestinal tract to ensure the full administration of the dose via the nasal cavity.
Te intranasal (i.n.) solution and nanoparticle formulations were administered using a microsyringe with an elastic top into the rat's nostril at a dose of 0.5 mg DHE/kg. Rats were given intravenous (i.v.) injections at a dose equal to 0.5 mg DHE/kg rat weight for the determination of absolute bioavailability. Testing for each formulation was done in triplicate as the approval number of the in vivo study was 3 rats for intravenous, 3 rats for intranasal solution, and 3 rats for intranasal nanoparticle formulation. Te nanoparticle formulation pH was maintained within the pH range of the nasal mucosa (pH 5.5-6.5) to avoid nasal irritation [46].
After administration of i.v. and i.n. DHE, the blood levels were determined by collecting 150 μL blood samples from rat tail veins stimulated by the milking mechanism.
Blood samples were collected at 0, 5, 10, 20, 40, and 60 minutes and then centrifuged at 6,000 rpm for 15 min. Plasma (60 μL) was frozen until the time of analysis.

Preparation of Plasma Samples by Liquid-Liquid
Extraction. Tertiary butyl methyl ether (TBME) (1 mL) was added to 60 μL plasma samples and vortexed for 5 min and then centrifuged at 13,000 rpm for 6 min. After separation, the organic layer was carefully removed and placed in a glass tube. Following the same procedure, another extraction was performed, and the resulting organic layer was also added to the glass tube. 200 μL of phosphoric acid 0.03 M was added to the tube and vortexed for 2 min. In a centrifugal evaporator, the TBME mixture was dried for 4 min under vacuum with no heat. Te remaining aqueous phosphoric acid layer was carefully collected into glass inserts and 60 μL aliquots were injected into the HPLC.

Statistical Analysis.
Pairs of groups were compared by Student's t-test. Diferences between groups were considered signifcant at p < 0.05. Values for all measurements are expressed as means ± SD.

Results
Chitosan nanoparticles were prepared using a modifed ionotropic gelation procedure by adding TPP aqueous solution to the chitosan solution. Figure 2 illustrates representative SEM imagies of CS-NPs. Nanoparticles loaded with 20, 30, or 40% DHE were obtained as described above. Characterization of particle size, loading, encapsulation efciency, polydiversity index, and zeta-potential is illustrated in Table 1. Te actual loading of DHE in the nanoparticles was measured and compared to the theoretical loading. Te encapsulation efciency for the 20, 30, or 40% loading was 95%, 83%, and 84%, respectively. Te average particle size ranged from 395 to 689 nm (Table 1).
Te integration of higher concentrations of DHE led to an increase in the size of the nanoparticles. Te results illustrated that the loading capacity and size of the nanoparticles were afected by the DHE concentration in the chitosan solution used to prepare the nanoparticles. Tese results agree well with the nature of DHE, which is positively charged under acidic conditions, leading to repulsion between positively charged groups in CS and DHE. Tus, the better encapsulation efciency and smaller nanoparticles loaded with 20% DHE were selected for further in vivo testing.
Te HPLC method was developed and validated with a calibration curve of R 2 � 0.999949. Te recovery of the area under the curve of aqueous samples and plasma was found to be higher than 90%. Double extraction was used to increase sensitivity and improve DHE detection limits. Te limit of detection was about 1.5 ng/ml, while the limit of quantifcation reached 1.7 ng/ml.
Te in vitro release profle of DHE chitosan nanoparticles under sink conditions is shown in Figure 3. It shows that about 53% of DHE was released in the dissolution media within 20 minutes and 82% ± 3.2 after 60 minutes. Tis release profle meshes well with acute migraine treatment strategies.
DHE was administered to rats intravenously at 0.5 mg/kg and i.n. solution and i.n. CS-NPs were also administered at 0.5 mg/kg. Te resulting blood plasma concentration of DHE versus time is shown in Figure 4. Intravenous administration resulted in signifcantly higher initial blood plasma concentrations than i.n. formulations (p < 0.05). At ten min following i.v. injection, DHE concentration peaked at 222.2 ng/mL±8 and then declined in a biphasic speed, frst rapidly, then tapering of slowly. Absolute bioavailability of DHE compared to i.v. administration was 53.2 ± 7.7% for i.n. solution and 82.5 ± 12.3% for i.n. DHE CS-NPs (Table 2). DHE was analyzed for pharmacokinetic parameters after i.v. and i.n. administration (Table 3). T max was reached at 40 minutes for both intranasal formulations, with C max of 213 ± 32 ng/mL (i.n. nanoparticles) and 161 ± 10.5 ng/mL (i.n. solution). Te i.v. DHE solution reached T max after only 10 minutes with a C max of 483.3 ± 69.9 ng/mL. Te half-life (T 1/2 ) of DHE in i.v. solution, i.n. nanoparticles, and i.n. solution was 14.0, 16.1, and 20.2 min, respectively.

. Discussion
Due to the demonstrated efectiveness of chitosan as a delivery vehicle [36,47], DHE was prepared as chitosan nanoparticles using ionotropic gelation of chitosan with TPP anions. Increased loading of DHE in nanoparticles produced a statistically signifcant (p < 0.05) increase in particle size. Terefore, loading additional DHE into chitosan nanoparticles increases the ionic charge ratio relative to chitosan,    as has been seen in the literature [47,48]. Te TPP polyanion favors the electrostatic interaction between the oppositely charged components. Te fnal DHE CS-NPs had a zetapotential of +26.5 mV and a polydiversity index of 0.438, which were in line with other chitosan nanoparticle formulations [49]. A higher zeta-potential indicates stability and a polydiversity index below 0.5 can indicate a similar distribution of particle size.
DHE was released rapidly from chitosan nanoparticles under in vitro sink conditions. Te hydrophilic nature of chitosan is the most likely variable afecting DHE release from the nanoparticles. Chitosan has been shown to improve the dissolution of poorly water-soluble drugs [50,51] by allowing an aqueous dissolution medium to access and dissolve the drug. Another factor could be that DHE was loaded into the CS-NPs near the surface. On the other hand, one might suggest that the main reason for this burst release of DHE from nanoparticles is its detachment from the polymer matrix due to repulsion.
Chitosan nanoparticles signifcantly enhanced in vivo DHE systemic absorption after i.n. administration (absolute bioavailability � 82.5 ± 12.3) when compared to i.v. dosing of DHE solution and to i.n. DHE solution (p < 0.05), although both i.n. solution and i.n. chitosan nanoparticles were absorbed quite rapidly.
When administering intranasal DHE in rats, limited nostril capacity limits the amount of drug absorbed through the nostrils, with 35%-40% absolute bioavailability [52], which is in the typical range of bioavailability for most intranasal DHE formulations [53]. Tus, chitosan nanoparticles as a delivery system for DHE enhanced absorption, most likely through the demonstrated ability of chitosan to adhere to mucosal tissues [45,[54][55][56][57][58][59]. However, chitosan's increased surface area [60] and the ability to open tight junctions [61] can also improve absorption. Rapid absorption through the nasal mucosa would be benefcial when attempting to quickly dose an uncooperative patient while avoiding injections, which often leads to patient noncompliance.
Te collected data showed that NP preparation has a superior bioavailability enhancement compared to conventional nasal solution preparation. In addition to   chitosan's mucoadhesive properties, its positive charge interacts with the anions on the mucus membrane surface to increase retention time, improving the drug absorption [62,63]. Te pharmacokinetic parameters ultimately demonstrate the concept of absolute bioavailability improvement. DHE CS-NPs' half-life (14.12 min) is higher than the solution (10.86 min), which indicates a longer therapeutic time. Studies have found the half-life of DHE after i.v. injection in beagle dogs to be 40.79 (0.5 mg)-70.13 min (1.0 mg) [64] and 104.42 minutes (2.0 mg i.v. injection) [65]. Te pharmacokinetics of DHE after i.n. and i.v. administration in rats was analyzed. Chitosan nanoparticles demonstrated potential for intranasal administration of DHE due to increased absorption compared to that of i.n. solution. Although both formulations reached maximum concentration within the same timeframe, CS-NPs followed a pattern of greater release. Rapid drug absorption is of particular beneft in migraines where patients require rapid relief or when a dose is missed. Furthermore, the preparation of DHE from chitosan nanoparticles appears to have overcome the slow onset of action which has been demonstrated with DHE [66]. Due to the sensitivity of the nasal mucosa, prophylactic nasal administration of DHE should be used with caution. Further clinical evaluation is warranted to determine the optimal dosing strategy of DHE CS-NPs for rapid relief of migraine.

Conclusion
Formulating DHE into chitosan nanoparticles has the potential to improve the systemic absorption of the drug by around 55%, resulting in a more rapid onset of action with a greater systemic bioavailability over DHE intranasal solution. Tis formulation could beneft migraine patients with improved pain relief, but it would need further clinical evaluation to illustrate its value.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.