Advanced Lipid-Based Nanocarriers for Naratriptan Hydrochloride: A Path to Improved Migraine Treatment

Advanced Lipid-Based Nanocarriers for Naratriptan Hydrochloride: A Path to Improved Migraine Treatment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Advanced Lipid-Based Nanocarriers for Naratriptan Hydrochloride: A Path to Improved Migraine Treatment Swati Lade This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6029615/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Migraine is a prevalent neurological disorder affecting a significant portion of the global population. It is characterized by frequent neurobiological or neurovascular disturbance, including heightened excitability of the central nervous system. Addressing these challenges, this study focuses on developing nanostructured lipids carriers (NLC) for intranasal drug delivery of naratriptan, aiming to provide a rapid and targeted treatment for migraine. The NLC was successfully formulated, characterized, and optimized using the Box Behnken Design (BBD). The optimized formulations underwent comprehensive characterization including assessments of % yield, encapsulation efficiency (%EE), particle size, Zeta potential, polydispersity index (PDI), surface morphology via scanning electron microscopy (SEM), crystallinity by X-ray Diffraction (XRD), and thermal behavior using differential scanning calorimetry (DSC), stability studies were conducted under varied conditions to evaluate formulation robustness. The optimized NLC formulation (NLCF2) demonstrated promising attributes with a % yield of 75.65±0.15 , % EE of 68.16±1.06 , particle size of 79.74±1.37, along with PDI of 0.315 , and Zeta Potential of -32.50. In-vivo pharmacodynamics studies using acetic acid-induced writhing and light/dark box models revealed significant efficacy of intranasal NLCs. Pharmacokinetic analysis, conducted using high-performance liquid chromatography (HPLC), demonstrated a 1.71-fold increase in drug concentration in the brain for the NLC formulation compared to the pure drug, underscoring enhanced bioavailability and brain targeting. The successful design and development of intranasal NLCs for migraine treatment represent a novel and effective approach to drug delivery. By facilitating rapid and targeted delivery, these nanocarriers have the potential to minimize systemic side effects and significantly improve the quality of life for individuals suffering from migraines. Drug Delivery Drug Discovery, Design, & Development Nanoscience Migraine Optimization Naratriptan solid-lipids NLCs Box Behnken Design Blood-Brain barrier Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 1. Introduction Migraine, a debilitating neurological disorder, is primarily characterized by recent episodes of headache accompanied by symptoms such as nausea, phonophobia, and photophobia. These episodes are often associated with significant cognitive and motor impairment, underscoring the profound impact of the disorder on a patient's quality of life. Despite extensive research, the intricate pathophysiological mechanisms underlying migraine remain incompletely understood, posing challenges to its effective treatment nevertheless, advancements in pharmacology have led to the development of a diverse array of therapeutic agents targeting various aspects of migraine pathogenesis(1–3). Naratriptan hydrochloride is a second-generation selective 5-HT1 receptor agonist and represents a notable advancement in migraine therapy. By selectively activating the 5 HT1B and 5-HT1D receptor subtypes, naratriptan exerts its therapeutic effects through dual mechanisms of constriction of dilated cranial arteries and inhibition of neurological inflammatory mediator release(4). Naratriptan, available exclusively in oral form at the recommended dose of 2.5mg, demonstrates an oral bioavailability of approximately 60% (5,6). Notably, it exhibits a six-fold higher affinity 5- HT1B receptors compared to sumatriptan underscoring its enhanced receptor specificity and therapeutic potential(7,8). Naratriptan can penetrate the intact blood-brain barrier (BBB) and exert its pharmacological effects on the trigeminovascular complex, particularly the trigeminocervical complex in the brainstem. However, achieving optimal therapeutic efficacy necessitates a drug delivery system capable of transporting the drug across the BBB in sufficient concentrations. Despite its clinical availability for over two decades, an efficient oral drug delivery system that specifically targets naratriptan to the brain has yet to be developed(9–11). The BBB, formed by tightly interconnected capillary endothelial cells, represents a significant obstacle in delivering therapeutic agents to the central nervous system (CNS). Effective drug delivery requires either surpassing or transiently disrupting this barrier without compromising its integrity. Innovative strategies, including nanoparticle-based carriers, ligand-mediated transport, or receptor-specific targeting, may offer a potential solution for overcoming these limitations and enhancing the CNS bioavailability of naratriptan, Developing such advanced delivery systems could transform the therapeutic landscape of migraine management by maximizing the efficacy of this potent triptan while minimizing systemic side effects.(12–14). The nanostructured lipid carrier’s drug delivery system have developed as a promising drug delivery system for overcoming the blood brain barrier and enhancing therapeutics efficacy in brain targeted treatments. (15). For minimizing the above-mentioned limitation associated with conventional regimen and achieve precise targeting, naratriptan-loaded nanostructured lipid carriers are proposed. The proposed formulation aims to achieve a high local drug concentration at a target site, thereby reducing the required dose and dosing frequency while significantly enhancing patient compliance(16–19). Nanostructured lipid carrier (NLCs), typically composed of a mixture of solid and liquid lipids in ratios ranging from 70:30 to 99:1, offers unique advantages. By blending these lipids in optimized proportion, the system provides increased capacity for effective drug encapsulation mostly the mixture of solid and liquid lipids from 70:30 to 99:1 offers a unique advantage. By blending these lipids in optimizing proportions, the system provides increasing spital capacity for effective drug encapsulation, in recent years, NLCs have emerged as superior drug delivery platforms due to their exceptional stability, biocompatibility, and the utilization of biodegradable excipients classified as generally regarded as safe. Additionally, their ability to provide a sustained drug release profile further underscores their potential as an advanced carrier system for therapeutic application (18,20,21). Preformulation studies are pivotal in providing comprehensive insights into the solubility, stability, permeability, and physicochemical properties of active pharmaceutical ingredients and excipients. These studies form the foundation for the rational design and development of pharmaceutical formulation. A critical component of preformulation involves evaluating the potential interaction between excipients and naratriptan, which can be either antagonistic or synergistic. Favorable interactions are particularly significant as they can enhance the pharmaceutical properties of naratriptan, including its stability, bioavailability, and pharmacokinetics profile. Conversely, negative interaction can compromise formulation performance underscoring the necessity of thorough investigation. The regulatory authorities emphasize stringent requirements for preformulation studies to ensure that the final formulation achieves optimal safety, stability, and maximum therapeutic efficacy. By identifying and optimizing key interactions during the pre-formulation phase, researchers can address potential challenges ensuring the development of robust and effective nanostructure lipid carrier formulations.(22–27). The concept of Quality by design has evolved intending to impart desired characteristics in the product by constraining the formulation variables and processing parameters. This study employs a Quality by Design (QbD) approach to optimize, prepare and characterize naratriptan-loaded nanostructure lipid carriers (NPNLC). These advanced nanocarriers have been developed as a brain-targeted delivery system to enhance naratriptan's central nervous system activity while minimizing its systemic exposure in peripheral tissues. This targeted approach amplifies the drug's antimigraine efficacy at lower doses and reduces associated side effects, offering significant advantages over conventional oral dosage forms such as tablets. By leveraging the QbD framework, the study ensures a systematic and robust development process, highlighting the potential of NPNLCs as an innovative and superior therapeutic modality for migraine management. (28–30) 2. Materials and methods 2.1 Materials Naratriptan hydrochloride was generously provided by USV Pharmaceutical Ltd. Mumbai, India Solid Lipid Excipients, including Gelucire ® 50/13 (Stearoyl macrogol-32 glyceride), Geleol ® Pellets (Glyceryl Palmitostearate), Compritol ® 888 (Glyceryl behenate), Glyceryl Monostearate (monostearic acid ester of glycerol), and Precirol ® ATO (Glyceryl stearate), were obtained as gigft sample from Gattefosse, France and Mohini Organic Pvt. Ltd., Mumbai, India. Medium chain triglyceride, including Capryol® 90 (propylene glycol monocaprylate), Miglyol® 808, Captex® 200P, Maisine® 35-1 (Glycerol monolinoleate), Campul ® MCM, and Capryol ® PGMC (Propylene glycol II monocaprylate were Provided by to ABITEC Mumbai and Mohini Organic Pvt. Ltd. Mumbai as gift Sample. Pluronic ® F127 was sourced from Sigma-Aldrich and sodium taurocholate from SD fine-Chen Limited, Mumbai. India. High-performance liquid chromatography (HPLC) grade Acetonitrile and water were purchased from SD Fine-chem Limited Mumbai. All other chemicals and reagents used during the study were of suitable analytical grade and were of suitable analytical grade and were used as received. 2.2 Solubility Assessment The solubility of naratriptan hydrochloride was determined using the water bath shaking method (Remi water bath shaker), Excess amounts of naratriptan were added to 2.0g of various oils, surfactants and co-surfactants. The mixtures were vortexed thoroughly to ensure proper mixing of naratriptan with the excipients and then equilibrated at 37 ±3 0 C in a water bath shaker. After 48 hours of incubation, the mixture were centrifuges at 12,000 rpm for 15 minutes to separate undissolved drug particles. The supernatant was filtered through a 0.45 µm membrane filter, and the filtrate was diluted with methanol. The concentration of naratriptan in the filtrate was analyzed using a UV-Vis spectrophotometer (UV-1800,Shimadzu,Japan)(31,32). 2.3 Compatibility studies Compatibility studies were performed to assess potential chemical interaction between naratriptan hydrochloride and the selected solid and liquid lipid material during the formulation of nanostructured lipid carriers (NLCs). Fourier Transform Infrared Spectroscopy (FTIR) was utilized for this purpose. FTIR spectra were recorded using a Bruker Spectrometer with the KBR pellet method over a wavelength range of 400-4000cm -1 .the scanning speed was set at 2 min -1 , with a normal slit width ensuring optimal resolution.(33). 3. Preparation and Experimental Design of naratriptan NLC Naratriptan nanostructured lipid carrier (NPNLC) were prepared using the melt-emulsification-ultra sonication process. The process includes separate processing of the lipid and aqueous phases. Glyceryl monostearate (solid lipids) and Capmul MCM (liquid lipid) in a 7:3 w/w ratio were accurately weighed and heated to 75 0 C which is 5 to 10 o C above melting point of the solid lipid, to prevent recrystallization during the process. Naratriptan was dissolved in the melted lipid phase. Simultaneously, an aqueous surfactant phase was prepared by dissolving Pluronic F 127 (nonionic surfactant) and sodium taurocholate (bile salt) in Millipore water. The surfactant solution was maintained at 75 0 C under continuous stirring using an electronic magnetic stirrer, Mumbai). The aqueous surfactant phase using a borosilicate glass syringe while stirring at 5-10 0 C above melting point of solid lipid The resulting coarse emulsion was subjected to probe ultrasonication (probe Ultrasonicator) in a water bath maintained at 750C for 15 minutes at 40 %amplitude. Finally, the prepared NPNLCs were cooled to room temperature on a magnetic stirrer, allowing the lipids to recrystallize and form the nanostructured lipid carriers. (29,34–37). Lyophilization of NPNLC formulation The NPNLC dispersions were lyophilized to obtain formulation in dry form of the formulation. Initially, the dispersions were frozen at −20ºC overnight, followed by lyophilization at −70ºC for 48 hours. The resulting lyophilized NPNLC powder was carefully collected and stored under appropriate conditions. Subsequently, the formulation underwent comprehensive physicochemical characterization and was subjected to in-vitro, ex-vivo, and in-vivo evaluation to assess its performance and therapeutic potential.(38) 3.1 Optimization using Box-Behnken Design. Preliminary studies were conducted to identify the key factor influencing the particle size, entrapment efficiency, and zeta potential of nanostructured lipid carriers (NLCs). These investigations highlighted the lipid concentration in the formulation as the most critical parameter. A randomized Box-Behnken Design (BBD) was employed to systematically evaluate the influence of three independent factors, each at two levels, on the dependent variables, namely particle size, entrapment efficiency, and percentage yield. The experimental matrix comprised 17 runs, including five center points to assess pure error, ensuring statistical robustness. The NLC formulation was prepared in a randomized sequence to minimize bias and ensure the reliability of the results.(39) The experimental design is summarized in Table 1. The excremental design, model fitting analysis, and all the regressions were performed using the Design Expert ® software version (stat-ease Inc., Minneapolis, MN)(40,40–45). Table 1: Formulation of Naratriptan according to Box-Behnken Design Std. Run Factor 1. Glyceryl monostearate (mg) 2. Capmul MCM (mg) 3. Pluronic F127 (%) 13 1 225 100 0.75 9 2 225 50 0.5 3 3 150 150 0.75 8 4 300 50 1 2 5 300 50 0.75 7 6 150 100 1 1 7 150 50 0.75 6 8 300 100 0.5 16 9 225 100 0.75 15 10 225 100 0.75 4 11 300 150 0.75 10 12 225 150 0.5 5 13 150 100 0.5 12 14 225 150 1 17 15 225 100 0.75 11 16 225 50 1 14 17 225 100 0.75 4. Evaluation and characterization of Naratriptan NLCs 4.1 Zeta potential and Particle size analysis Determination The Zeta potential and average particle size of formulated nanostructure lipid carriers (NPNLC) were evaluated in HPLC grade water. Mesurements were performed in triplicate using photon correlation spectroscopy on Zetasizer Nano ZS90 (Malvern Instruments, UK) opening in automatic mode. The analysis were conducted at 25 0 C with a detection angle of 900, ensuring precise and reproducible results.(46,47). 4.2 % Entrapment efficiency (EE) The formulated nanostructured lipid carriers (NPNLC) were isolated from the dispersion medium by centrifugation at 15,000 rpm for 20 minutes at 40C. The clear supernatant was carefully decanted and analyzed for the free drug content using UV-visible spectrophotometer at the maximum absorbance wavelength (λ max). A placebo NLC (formulated without the drug) was subjected to identical centrifugation conditions (15,000 rpm, 20 minutes at 40 C) and served as the blank. A standard calibration curve for Naratriptan hydrochloride was constructed by plotting absorbance against concentration (x- axis) to check the free drug content.(48,49). The percentage of drugs entrapped was calculated by using the following equation: 4.3 In vitro Drug studies: The in vitro drug release profile of all the formulated NPNLC batches was determined using a USP Type II dissolution apparatus (TDT-08L Electrolab, India). Drug-loaded NLCs, equivalent to 5mg of Naratriptan hydrochloride,were redispersed in 2 mL of phosphate buffer (pH 6.4) and enclosed in a pre-soaked dialysis membrane bag. The dialysis bag was securely tied to the paddle of the dissolution apparatus and immersed in 500 ml phosphate buffer (pH 6.4), serving as the dissolution medium. Prior to experimentation, the dialysis membrane was soaked in deionized water for one hour to ensure optimal permeability. The dissolution medium was maintained at a temperature of 37°C ± 1°C and agitated at a speed of 100 rpm respectively. At predefined time intervals, 5 mL aliquots were withdrawn and immediately replace with an equal volume of fresh phosphate buffer (pH 6.4) to maintain the sink conditions. The percentage cumulative drug release was estimated by measuring the UV absorbance of the aliquots, at λmax = 226 nm, against the blank dissolution medium as the reference. All experiments were performed in triplicate, and results are presented as mean % drug release ± standard deviation. The drug release mechanism was elucidated by fitting the in vitro release data to various release kinetic models, including zero order, first order, Higuchi model, Hixson-Crowell, and Korseymere Peppas models, using appropriate mathematical software. (41,42) . 4.4 Fourier- Transform Infrared (FTIR) Spectroscopy The FTIR spectra of lyophilized optimized drug-loaded NPNLC batch, the physical mixture of Naratriptan hydrochloride and excipients in proportions used for fabricating NPNLC, Placebo nanoparticles (formulated without drug ), and pure Naratriptan hydrochloride were recorded using an FTIR spectrophotometer (Perkin Elmer, USA) in the frequency range of 400–4000 cm−1. The KBr pellet method was employed to observe the potential chemical interactions between the pure drug and excipients within the formulation. (50,51) . 4.5 Powder wide-angle X-ray diffraction The XRD patterns of pure Naratriptan hydrochloride and optimized NPNLC formulation were analyzed using an X-ray Diffractometer (Rigaker Geiger flex, Japan). Prior to analysis, the NPNLC formulation was lyophilized to ensure accurate characterization. Diffractograms were recorded from the initial angle 2θ =100 to the final angle of 400 with a CuKα radiation source. Samples were mounted in the glass sample holders and scanned over a temperature range of 20°C to 80°C with a scan rate 2o/min, under operating conditions of 35kV and 40 mA current. (52,53) . 4.6 Differential Scanning Calorimetry (DSC): Differential Scanning Calorimetry (DSC) thermal analysis was performed to evaluate the physical state and thermal behavior of the pure Naratriptan Hydrochloride, and the optimized NPNLC formulation. Thermogram were recorded on DSC analyzer (TA Instruments DSC SDT Q600), to determine the physical nature of the drug and the polymers/carriers in the formulation. Approximately 4–5 mg of each sample was accurately weighed, sealed in aluminum pans, and hermetically crimped to prevent moisture loss and ensure uniform heat distribution. The samples were scanned over a temperature range of 25–400◦C at a heating flow rate of 10◦C/min under a continuous nitrogen purged maintained at a flow rate of 100 mL/min. The data was analyzed using the Universal Analysis 2000 software package (SDT Instruments). (40,54–57) . 4.7 Scanning electron microscopy (SEM) The surface morphology study was carried out to examine the surface morphology and shape (Jeol Japan 6000). The surface morphology of the optimized NPNLC formulation were analyzed using a Scanning Electron microscope (SEM, JEOL JSM-6000 Japan). SEM employs a focused beam of electrons, rather than light, to generate highly magnified images by interacting with the sample surface, leading to the emission of secondary electrons and X-rays. Prior to imaging, the samples were carefully mounted onto metal stubs using a double-sided adhesive tape and sputter coated with a thin conductive layer under a vacuum. The analysis was performed at various magnifications, at an acceleration voltage of 15 kV, and with a working distance maintained at 20 μm. Microphotographs were taken at different magnifications, and surface morphology was examined at a higher magnification. (50,52) . 4.8 Ex vivo drug release study An ex vivo drug diffusion study was conducted using freshly excised sheep nasal tissue to evaluate the drug release characteristics of Naratriptan hydrochloride and its nanostructed lipid carrier (NPNLC) formulation. Freshly excised sheep nasal tissue was collected from a local slaughterhouse, immediately immersed in freshly prepared phosphate buffer solution (pH 6.4), and transported to the laboratory. The bony cartilage was carefully removed from the mucosal membrane; and the nasal tissue was thoroughly washed and stabilized in a phosphate buffer (pH 6.4) before experimentation. The tissue was mounted between the donor and receptor compartments of a Franz diffusion cell under a phosphate buffer solution (pH 6.4). In the donor compartment 1mL of Naratriptan Hydrochloride pure drug solution (5 mg/mL) was introduced onto the nasal tissue, while the receptor compartment was filled with 50 mL of phosphate buffer solution (pH 6.4). The stirring speed was maintained at 200 rpm using a magnetic stirrer, and the temperature was kept constant at 37±50C to mimic physiological conditions. For NPNLC formulation, lyophilized NPNLCequivalent amount to 5 mg of Naratriptan was reconstituted in 1 mL of phosphate buffer solution (pH 6.4) and applied to the nasal tissue in the donor compartment. Aliquot 2mL of Samples at predetermined time intervals (0.5, 1, 2, 4, 6, 8, 10, 12, and 24 hours) were withdrawn from receptor compartment and immediately replaced with an equal volume of fresh phosphate buffer (pH 6.4) to maintain sink conditions. The amount of drug diffused across the nasal tissue was analyzed using a UV–visible spectrophotometer (Wenser) at a wavelength λmax 226 nm. The percentage of drug diffusion was calculated using a pre-established standard calibration curve for Naratriptan hydrochloride. (55–59) . 4.9 Stability Investigation The stability of the lyophilized NPNLC formulation was evaluated by storing the samples in a screw-capped glass bottles covered with aluminum foil to protect them from light exposure. The samples were maintained under two storage conditions: refrigerated temperature (4-8o C) and a room temperature (250C) for a period of 1-6 months. At predetermined intervals, the formulations were analyzed for percent encapsulation efficiency (% EE) and Drug content. The results were compared with a freshly prepared NPNLC formulations. (50,60) . 5. In vivo Studies Adult male Swiss albino mice (25-30 g) and adult male Wistar rats (200-250 g) were utilized in the present study. The animal Activity protocols for the optimized formulation were approved by Dr. D. Y. Patil Institute of Pharmaceutical Sciences & Research, Sant Tukaram Nagar, Pimpri, Pune 411018 with Institutional Animal Ethical Approval No DYPIPSR/IAEC/OCT/21-22P-19, in compliance with the guidelines established by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. Prior to experimentation, animals were acclimatization to the laboratory conditions for at least one week. They were housed under a controlled environment with a 12 hour light/dark cycle, at a temperature of 25° ± 1°C. Standard laboratory diet and water were provided ad libitum throughout the study period. 5.1 Pharmacodynamic study The optimized nanostructured lipid carrier (NLC) formulation was evaluated for its efficacy in migraine management using established behavioral models. The acetic acid-induced writhing test was employed to assess hyperalgesia, while the light /dark box test was utilized to evaluate photophobia, two hallmark symptoms of migraine. These studies were conducted Swiss albino mice, ensuring relevance to migraine symptomatology and their therapeutic potential. (60) . 5.1.1 Acetic acid-induced writhing test: Adult male Swiss albino mice were randomly assigned to four different groups (n=6 per group) :control (normal saline), standard drug (NP), placebo formulation (NLC), and the optimized test formulation (NPNLC). The drug and test formulations were orally administered after dispersion in deionized water. Hyperalgesia was induced by intraperitoneal injection of Acetic acid solution (0.3%, 10 mL/kg). Five minutes after acetic acid administration, the number of abdominal writhes was counted over a 10 minute observation period. 5.1.2 Light/dark box model: Photophobia, a hallmark symptoms of migraine, was evaluated in Swiss male albino mice in terms of light aversiveness using light and dark box models (61) . The light/ dark box apparatus consisted of a rectangular box (60x60x45 cm3), divided equally into two compartments connected by an opening (7x7 cm2). The light compartment (LC) was illuminated by a 60w bulb and left uncovered from the top, while the dark compartment was completely painted black and fully enclosed on all sides. Mice were assigned to treatment groups, including NPNLC, Placebo (NLC), and control and were administered their respective treatments for seven consecutive days. On the experimental day, each of the animals was placed in a light compartment and observed for 5 minutes. The time spent in the light compartment was recorded and expressed as a percentage of the total observation period, calculated as follows: 5.1.3 Determination of Brain Uptake Potential Two of the output responses for formulation optimization were drug concentration in the brain and the AUC brain/AUC plasma 2 hours after drug administration. Male Wister rats were divided into 4 groups (n=3): normal saline (control), std. (5 mg/kg by nasal route NP), placebo (NLC), and formulated formulation (5 mg/kg by nasal route NPNLC). Prior to the trial, animals were fasted overnight with no access to water. The medicine, placebo, control, and test formulation were administered through the nasal route to the complete in vivo trial after 0.5ml blood was administered in heparinized tubes at different times of 30, 75, 135, 225, and 240 minutes. Samples were centrifuged at 8000 rpm for 10 minutes, and the plasma were separated and stored at 20°C till the next processing and analysis. Following blood samples collection, the animals were sacrificed by decapitation under deep anesthesia at various intervals of 30, 75, 135, 225, and 240 minutes. The brain was quickly extracted, weighed, and blotted to remove excess surface blood. The brain tissue was homogenized in 0.01 M phosphate buffer (pH 6.4), then centrifuged at 8000 rpm for 10 minutes. The supernatant was stored at - 200C fo subsequent processing and analysis. In the present study, chromatographic separation was performed using an HPLC system (prominence version 1.24 Sp1, Shimadzu Corporation, Japan), controlled by LC solution software. Before HPLC analysis, plasma and brain samples were subjected to liquid-liquid extraction. a 200µl aliquot of plasma or brain sample was treated with 20µl of standard solution (100 µl/ml and 200µl/ml) and 2M sodium hydroxide (200 µL). After vortexes mixing for 1 minute, 2 ml of ethyl acetate was added, followed by shaking for 2 minutes, and centrifuged at 10000 rpm for 15 min. at 100C. the extraction procedure was repeated twice. The supernatant (organic phase) was collected, evaporated to dryness, and reconstituted with 200 µl mobile phase. The drug was quantified using a validated HPLC method applied to plasma and brain extracts. Prior to injection into HPLC analysis, sample (20 μl) were filtered through a 0.22 µm syringe filter. HPLC analysis was conducted using a Shimadzu Corporation system equipped with Photodiode array (PD) detector and symmetry® C18 column (5µm particle size, 4.6 mm × 250 mm). The mobile phase consisted of a mixture of acetonitrile and 0.05 M potassium dihydrogen phosphate (80:20, v/v), with 10% acetonitrile and 0.1 % triethylamine, pH adjusted to 3.8 with o-phosphoric acid. The flow rate was set at 1 ml/min, the total run time was 12 minutes, and UV detection was performed at λ max 226 nm. Brain targeting potential was assessed by determining the drug concentration in both brain and plasma. The ratio of AUC (brain) /AUC (plasma) was calculated after 2 hours of drug administration using the trapezoidal method. The pharmacokinetic parameter was estimated using PK solvers add-in program for the Microsoft Excel. Key parameters, such as maximum plasma concentration (Cmax) and the time to reach maximum plasma concentration (T max) were directly obtained from the plasma concentration versus time curve. The area under the curve (AUC) was calculated for further analysis. Relative bioavailability was calculated by (F) = AUC test/AUC reference x100. 6. Statistical analysis Statistical analysis was conducted using GraphPad Instata software (version 3.06). Results are presented in terms of mean ± standard deviation (SD) of the mean. One-way analysis of variance (ANOVA) was employed to correlate the statistical significance of the results, followed by the post hoc Tukey-Kramer test to determine the significant differences between groups. The statistical significance was set at a P-value less than 0.05. 7. Result and discussion In this study, the randomized Box-Behnken Design, a quadratic randomized response surface methodology, was used to optimize the formulation for the optimization of formulation for seventeen experimental runs, ensuring a systematic and robust approach to parameter evaluation. The effects of key factor including the amount of solid lipid (Glyceryl monostearate) (A), liquid lipid (Capmul MCM) (B), and surfactant concentrations (C), on critical quality attributes (CQAs) of the prepared NLCs were systematically evaluated. The CQAs assessed namely, percentage yield (R1), entrapment efficiency (% EE) (R2) and particle size (R3). The mean particle size across all the formulation batches ranged from 79.74 ± 1.37 nm to 185.58 ± 0.43 nm, the percentage yield varied between 60.54 ± 0.74% to 75.65 ± 0.15%, while the entrapment efficiency of the formulation ranged from 60.12 ± 2.1% to 75.65 ± 0.55%. The responses were analyzed using the general polynomial equations, and Analysis of Variance (ANOVA) was performed utilizing Design Expert software. Statistical parameters, including degrees of freedom (df), sum of squares, mean sum of squares, and Fischer’s value (F-value) were evaluated to value model performance. The statistical significance of the model was determined based on the F-value, P-value, correlation coefficient (R 2 ), and adjusted correlation coefficient (adj R 2 ). The regression equations derived for the response variables are presented as follows: (Table 2 ) The regression-coded equations derived for the response variables are as follows: Final Equation in Terms of Coded Factors Percentage Yield (X 1 ) = + 64.39 + 3.57 A + 2.34 B + 4.52 AB + 3.53 A² ( 1 ) Entrapment Efficiency (X 2 ) = + 62.25 + 3.13A + 3.7B + 4.66A² ( 2 ) Particle Size (X 3 ) = + 145.32–31.87 A -15.96 B -5.06 C -18.84 AB -13.71 AC -5.93 BC -9.82 A² +12.88 B² -11.15 C² ( 3 ) Where, A = Conc. of Glyceryl Monostearate B = Conc. of Capmul MCM C = Pluronic F127 X1 = Percentage Yield X2 = Entrapment Efficiency X3 = Particle size From Eq. (1) it can be conferred that an increase in concentration of Glyceryl monostreate (A) positively influences the percentage yield (X 1 ) and an increase in the concentration of both Glyceryl monostearate (A) and Capmul MCM (B) lead to an increase in the percentage yield. In contrast, the interaction between Glyceryl monostearate (A) and the Pluronic F127 (C) resulted in a reduction in the percentage yield of formulations. From Eq. (2) it is evident that the entrapment efficiency of a formulation increases with the concentration of all three parameters (Glyceryl Monostearate (A), Capmul MCM (B), and Pluronic F127 (C)). However, the interaction between Glyceryl monostearate (A) in combination with the Pluronic F127 (C) leads to decreases the entrapment efficiency when the concentrations of both are decreased. Eq. (3) revels that the particle size of NPNLC decreases with a reduction in the concentration of the Glyceryl monostearate (A), Capmul MCM (B), and Pluronic F127 (C). Apart from this if we double the concentration of Capmul MCM the particle size increases. (Fig. 1) Table 2 Regression Analysis Regression Analysis Response The regression equation for coded factors R 2 Adjusted R 2 Adeq. Precision % Yield Yield = + 64.39 + 3.57 A + 2.34 B + 4.52 AB + 3.53 A² 0.8371 0.6277 7.2089 Adequate precision value, which measures the signal-to-noise ratio, indicates the reliability of model in predicting responses. A ratio greater than 4 is considered desirable, and your ratio of 7.2089 confirming that the model is suitable for navigating the design space effectively. %EE Entrapment Efficiency = + 62.25 + 3.13 A + 3.77 B + 4.66 A² 0.8721 0.7076 8.8379 Adequate Precision value, measures the signal-to-noise ratio. A ratio greater than 4 is considered desirable. Obtained ratio of 8.838 suggests a robust signal, indicating that the model is suitable for navigating and optimizing the design space. Particle size Particle Size = + 145.32–31.87 A -15.96 B -5.06 C -18.84 AB -13.71 AC -5.93 BC -9.82 A² +12.88 B² -11.15 C² 0.9882 0.9729 28.88 Adequate Precision, which measures the signal-to-noise ratio, serves as an indicator of the models reliability n predicting responses. A ratio greater than 4 is acceptable, and ratio of 28.885 indicates an adequate signal. This model can be used to effectively navigating and optimizing the design space. 7.2 Lipid Screening The various solid and liquid lipids are used for the screening of lipids in NLC production based on the solubility of a drug in lipid selection of lipids are done. In this study, we found that the drug naratriptan showed a higher amount of solubilization in Glyceryl monostearate and Campul MCM. As more solubility of a drug in these lipids, we selected this lipid for feature development of NLC. The selection of surfactant was done based on the Hydrophilic lipophilic balance (HLB scale) and the physicochemical property which is most suitable for the nose-to-brain drug delivery system. (Fig. 2 ) 7.3 Physicochemical Characterization of Naratriptan NLC 7.3.1 Desirability The primary objective of pharmaceutical formulation optimization is to discover the stages of the variable that most significantly influence the selected responses and to determine the optimal levels of these variables from which to develop of robust product with superior-quality attributes. In this study, all the measured responses affecting product quality were considered during the optimization process. The optimization criteria were set to maximize both percentage yield and entrapment efficiency (EE), while minimizing particle size. The graph shows that the ideal suitability of the NLC composition was 0.983 which is closely related to the ideal value, indicating a strong alignment between the predicted parameters and desired parameter values. (Fig. 3) The predicted formulation shows a composition identical to that of NLCF 2, further validating the accuracy of the optimization model. (Table 3 ), Table 3 Characteristics of optimum formulation SN Objects Glyceryl monostearate Capmul MCM Pluronic F127 % Yield %EE Particle size (nm) Desirability 1 Predicted 300 50 1 70.79 66.86 80.98 0.983 2 Actual 300 50 1 75.65 68.16 79.74 7.3.2 Fourier -Transmission Infrared (FTIR) Spectroscopy The FTIR spectrum of pure drug NP exhibited a distinct peak at 2918.93 cm -1 , corresponding to the CH stretching vibration, a band at 1146.74 cm -1 attributed to C = O stretching, and a peak at 1064.57 cm -1 corresponding to S = O stretching vibration band. (Table 4 ), The FTIR spectrum of NP formulation represented all the characteristic peaks of both the drug and the excipients, thereby confirming the absence of any chemical interaction between them. (Fig. 4 , 5 ) Table 4 Interpretation of FTIR Graph SN Name of Functional Group Wave number cm-1 1 C-H Stretching asymmetric 2918.93 2 C-H stretching symmetric 2851.52 3 C = O Stretching 1146.74 4 C-N stretching 1646.74 5 -S = O stretching 1064.57 7.3.3 Differential Scanning Calorimetry (DSC) The DSC thermogram presented in Fig. 6 reveals critical insights into the melting point and the crystalline or amorphous nature of naratriptan hydrochloride and its lipid carriers. A sharp endothermic peak observed at 226-236 0 C in the thermogram of pure naratriptan hydrochloride indicates its crystalline structure and characteristic melting point. This sharp endothermic peak of naratriptan is absent in the thermogram of naratriptan loaded nanostructured lipid carriers (NPNLCs), indicating that either naratriptan is completely solubilized in the lipid matrix or has transitioned into an amorphous form upon incorporation into the carrier system. Furthermore, the DSC profile of NLCs was investigated and analysis with Glyceryl monostearate and portion amount Campul MCM in the ratio of 70:30 used for the NLCs formulation, indicative of reduced crystallinity of Glyceryl monostearate which shows broadening of the thermal peak. This change in thermal behavior highlights the effective encapsulation of naratriptan within the lipid carriers. (Fig. 6 ) 7.3.4 Percentage yield (% yield), % Entrapment Efficiency (%EE), and Particle size Percentage yield (% yield), % Entrapment efficiency (% EE), and Particle size are shown in Table 5 . Table 5 Validation of predicted and experimental values of NLC formulation SN Response Experimental Value ± SD Predicted Value % Relative Error 1 % Yield 75.65 ± 0.15 70.79 6.86 2 %EE 68.16 ± 1.06 66.86 1.94 3 Particle size(nm) 79.74 ± 1.37 80.98 1.53 7.3.5 Result of Particle Size of NLCF2 The average particle size of NTP-NLC was measured using dynamic light scattering (DLS), Malvern zeta sizer (Malvern zeta sizer, Worcestershire, UK) from SAIF RGPV, Bhopal. The particle size of optimized formulations is as follows for NLCF2 is 79.74. (Fig. 7) The figure indicates the intensity of size distribution here the contributions in terms of % intensity are shown as a function of Particle diameter the Z value was recorded as 79.74 with a width of 17.45. 7.3.6 X-ray diffraction study The X-ray diffractogram (Fig. 8 ) shows a very high intensity, depicting the crystalline nature of pure naratriptan hydrochloride (NP). In contrast, the X-ray Diffractogram of lyophilized formulation NPNLC shows a significant reduction in peak intensity.This decrease suggests successful incorporation of drug into the lipid carrier system, accompanied by a slight reduction in its degree of crystallinity. These results show that, potentially enhancing its solubility and bioavailability. 7.3.7 Scanning electron microscope (SEM) The optimized naratriptan formulations' spherical shape was confirmed by an SEM photomicrograph. The particles had moderate uniformity and were all discrete entities with no aggregation. They were racially homogenous to smooth surfaces and had no rupture and the size was discovered to be within the limit. (Fig. 9 ) 7.3.8 In vitro Drug release The in vitro drug release profile of naratriptan from NLC formulation was evaluated using the dialysis bag method and compared with a Naratriptan drug solution. The study was conducted in a phosphate buffer (pH 6.4) due to partial solubility of drug in the buffer solution, as reported in literature. The results demonstrated that64.36% of the drug was released from the pure naratriptan solution within 2 hours, whereas the NPNLCs formulation exhibited a sustained release pattern with a cumulative drug release of 99.12% over 24 hours Fig. 10 . It was concluded that the released rate from nanostructured lipid carrier could be significantly prolonged the release of Naratriptan. Furthermore, it was observed that the drug release rate decreased with an increase in NLC particle size. The in vitro release data were fitted to various kinetic models, such as zero order, first order, and Higuchi, Korseymere Peppas, and the Hixson-Crowell models, with correlation coefficients (R 2 ) of 0.9708,0.9728, 0.9901, 0.9850 and 0.9711 respectively, indicating that the release mechanism is best described by Higuchi model. 7.3.9 Ex-vivo diffusion studies Ex-vivo diffusion tests on sheep nasal mucosa for NP solution and NPNLC preparation were carried out. After 24 hours at pH 6.4, the drug's ex-vivo release for NPNLC was found to be 90.12 ± 1.21%. Whereas, the NP solution demonstrates 76.28 ± 1.56% drug release; it persists in protonated form at nasal mucus layer pH (5.5–6.5) and is transferred via tight junctions started by sodium taurocholate. Increase permeation through nasal mucosa as a result of paracellular transport across tight junctions. (Fig. 11 ) 7.3.10 Stability Studies of NPNLC The stability of the optimized NPNLC formulation was evaluated over 6 months under three different storage conditions like 4 ± 2 0 C, 25 ± 2 0 C / 60 ± 5% RH, and 40 ± 2 0 C /75 ± 5% RH. Drug content and entrapment efficiency were noted every time we increased temperature and time interval, implying also that NPNLC composition remains constant for more than 6 months, as shown in Table 6 . These findings confirm the robust stability profile of the formulation under varied environmental conditions, ensuring its suitability for therapeutic application.( 62 – 65 ) Table 6 Stability studies of NPNLC SN Parameter Optimized Formulation NLCF2 Temperature (oC) Initial 4 ± 2 oC 25 ± 2 oC/60 ± 5% RH 40 ± 2oC/75 ± 5% RH 1 % EE 73.25 ± 0.68 72.91 ± 0.44 72.05 ± 1.09 71.00 ± 0.88 2 Drug Content 97.60 ± 0.48 96.54 ± 0.45 96.66 ± 0.12 97.19 ± 0.09 7.3.11 Pharmacodynamic study: Intranasal administered NPNLC reaches the brain cell after crossing BBB through the endocytic process. 7.3.11.1. Acetic Acid Induced writhing test. The pharmacological efficacy of the NPNLC formulation was evaluated using an acetic acid-induced writhing assay in mice, characterized by abdominal constrictions and hind limb stretching. Figure 12 shows a significant reduction in the amount of writhing in the formulation- treated group compared to the control and pure NP groups. A placebo NLC group was used to assess the potential influence of excipients on the pharmacological activity, and no significant effect was observed compared to the control group. Literature suggests that the Naratriptan Drug has to navigate across BBB to reach the somatic afferent terminals of the spinal cord, where it produces a hyperalgesic effect. These findings indicate that the NPNLC formulation effectively permeated the BBB, modulating the trigeminovascular system and reducing the abdominal writhing’s and it is used for enhanced therapeutic efficacy. 7.3.11.2. Light /dark box model The light/dark box model was employed to assess photophobia, which is one of the most common symptoms associated with migraine characterized by an allodynic response to light. Figure 13 demonstrates that the optimized NPNLC formulation significantly increased the time spent by Swiss albino mice in the lit compartment compared to the control, placebo and pure NP groups. The placebo NLC group showed no significant effect compared to the control group, indicating that the excipients did not influence the observed pharmacological activity. The result concluded that the NPNLC formulation successfully crossed the blood-brain barrier (BBB) and modulated the activity of the trigeminal nucleus caudalis, ultimately enhancing light tolerance and reducing photophobic responses in treated animals. ( 66 – 69 ) 7.3.11.3. Determination of Brain Uptake Potential The developed and validated HPLC technique effectively quantified the drug concentrations in both brain and plasma samples. Result demonstrated a 1.71-fold rise as in drug which obtained a brain from NPNLC once compared with the pure drug. Furthermore, AUC brain / AUC plasma proportion was determined to also be 2.13 and 1.25 after post two hours of administering drugs in the NPNLC and NP, respectively. (Fig. 14 , 15 , 16 , 17 ). these findings underscore the enhanced brain-targeting capability of NPNLC formulation.( 70 – 73 ) 8. Conclusion The present study successfully optimized Naratriptan nanostructured lipid carriers (NPNLC) using a Box-Behnken design, focusing on key parameters such as particle size, entrapment efficiency, and % yield. This optimization approach provided valuable insights into the interactions between formulation parameters, which can be explained by particle size, entrapment efficiency, and % yield. The resulting optimized formulation was assessed for the pharmacodynamic study by using acetic acid writhing and light/dark models by comparing with a pure drug which shows remarkable results by comparison. The Quality by Design QbD approach thus proved to be a robust strategy for improving the brain-targeting potential of NPNLC, ultimately leading to enhanced anti-Migraine activity. 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3","display":"","copyAsset":false,"role":"figure","size":83304,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesirability\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6029615/v1/6a458b09a1044e33161599de.png"},{"id":76441335,"identity":"df8ff720-d7d8-4bd5-8f56-8aa6e281380d","added_by":"auto","created_at":"2025-02-17 08:17:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":26171,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFTIR spectra of Naratriptan\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6029615/v1/4c4d0eccac4a8dbffd29f3a0.png"},{"id":76442540,"identity":"05df6c04-de90-4973-80d5-909c9aeb856d","added_by":"auto","created_at":"2025-02-17 08:25:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":26555,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFTIR spectra of Naratriptan and All Excipients\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6029615/v1/f4565d974ae2cc69ab5845f3.png"},{"id":76442545,"identity":"aeb80346-09e1-4b83-9472-5cd87f11b204","added_by":"auto","created_at":"2025-02-17 08:25:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":83551,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDSC of Pure drug and NPNLC\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6029615/v1/cc99e06ea63a84a0f71f785c.png"},{"id":76441352,"identity":"ea1d810b-bdad-4cf3-a478-f2b136dcae6a","added_by":"auto","created_at":"2025-02-17 08:17:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":108623,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eParticle Size of NLCF2\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6029615/v1/3845fdf3cf8e6f6227d5cea8.png"},{"id":76442548,"identity":"c43744c9-e8c1-4faf-867e-006ace1ae66a","added_by":"auto","created_at":"2025-02-17 08:25:53","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":103769,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eXRD of Pure Drug and NPNLC\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6029615/v1/9e1dc7003641cbaaf2a340fe.png"},{"id":76442851,"identity":"cbee2c18-52bb-498f-84bc-1629927c5955","added_by":"auto","created_at":"2025-02-17 08:33:53","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":248266,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM image of NPNLC\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6029615/v1/c0bf8c645b4d6770fa636162.png"},{"id":76442553,"identity":"367fe034-5123-45c3-a287-a12eb48480de","added_by":"auto","created_at":"2025-02-17 08:25:54","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":164508,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro drug release of formulations\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6029615/v1/d10360ac995739b137ec188a.png"},{"id":76444272,"identity":"a74f0370-aa2d-47da-98bb-6fcdb6aa4407","added_by":"auto","created_at":"2025-02-17 08:41:53","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":34698,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEx vivo diffusion study series 1 (NPNLC), Series 2 (Pure NP Solution)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-6029615/v1/b42d07b7c5583cafd08e8bea.png"},{"id":76442542,"identity":"f3c9e9f0-661e-4109-a365-662c86198691","added_by":"auto","created_at":"2025-02-17 08:25:53","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":24343,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePharmacodynamic study by Acetic acid-induced writhing test\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-6029615/v1/2e4aeafe69b02eed9970070a.png"},{"id":76442555,"identity":"7658594f-656a-4476-a791-9b3514e91b2f","added_by":"auto","created_at":"2025-02-17 08:25:54","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":22566,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePharmacodynamics study by time consumed by the animal in light compartment of light/dark box model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e***P\u0026lt;0.001significantly different from all groups one-way ANOVA followed by Tukey-Kramer test\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-6029615/v1/cf7ded4cc0130307b75b3761.png"},{"id":76444472,"identity":"4b69ea27-d0da-483b-8587-7b1ebdbff867","added_by":"auto","created_at":"2025-02-17 08:49:53","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":17745,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of Cmax value of NPNLC and NP in Plasma\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-6029615/v1/37bac74003f1e3aa85e19d9f.png"},{"id":76441364,"identity":"242cd27f-3b65-43f8-8a76-25b090bb24f4","added_by":"auto","created_at":"2025-02-17 08:17:54","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":26029,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEstimation of NPNLC and NP in plasma\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"15.png","url":"https://assets-eu.researchsquare.com/files/rs-6029615/v1/f8eb82d4e44e6eab2f081ae4.png"},{"id":76441367,"identity":"f24a1862-f9cf-4fd7-ad43-32c07298d507","added_by":"auto","created_at":"2025-02-17 08:17:54","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":15709,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of C max values of NPNLC and NP in Plasma and Brain homogenate\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"16.png","url":"https://assets-eu.researchsquare.com/files/rs-6029615/v1/5226d51530e08914e488a3f8.png"},{"id":76441358,"identity":"2b338060-bf45-4aee-807e-131f64f875d7","added_by":"auto","created_at":"2025-02-17 08:17:53","extension":"png","order_by":17,"title":"Figure 17","display":"","copyAsset":false,"role":"figure","size":28413,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEstimation of NPNLC and NP in Brain homogenate\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"17.png","url":"https://assets-eu.researchsquare.com/files/rs-6029615/v1/e23b1cb43795c1a5c3319f9d.png"},{"id":76674827,"identity":"7777b6bb-a208-4ae5-b9e2-0c2c692c8856","added_by":"auto","created_at":"2025-02-19 14:16:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3503869,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6029615/v1/d97f2ee2-3560-4893-9b69-1a2be5bba16f.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eAdvanced Lipid-Based Nanocarriers for Naratriptan Hydrochloride: A Path to Improved Migraine Treatment\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMigraine,\u0026nbsp;a\u0026nbsp;debilitating\u0026nbsp;neurological disorder,\u0026nbsp;is primarily characterized\u0026nbsp;by\u0026nbsp;recent episodes of headache accompanied by\u0026nbsp;symptoms\u0026nbsp;such\u0026nbsp;as nausea,\u0026nbsp;phonophobia, and\u0026nbsp;photophobia. These episodes are often associated with significant cognitive\u0026nbsp;and\u0026nbsp;motor\u0026nbsp;impairment, underscoring the profound impact of the disorder on a patient's quality of life.\u0026nbsp;Despite extensive research, the intricate pathophysiological mechanisms underlying migraine remain incompletely understood, posing challenges to its effective treatment nevertheless, advancements in pharmacology have led to the development of a diverse array of therapeutic agents targeting various aspects of migraine pathogenesis(1–3).\u003c/p\u003e\n\u003cp\u003eNaratriptan hydrochloride is a second-generation selective 5-HT1 receptor agonist and represents a notable advancement in migraine therapy. By selectively activating the 5 HT1B and 5-HT1D receptor subtypes, naratriptan exerts its therapeutic effects through dual mechanisms of constriction of dilated cranial arteries and inhibition of neurological inflammatory mediator release(4). Naratriptan, available exclusively in oral form at the recommended dose of 2.5mg, demonstrates an oral bioavailability of approximately 60%\u0026nbsp;(5,6). Notably, it exhibits a six-fold higher\u0026nbsp;affinity 5- HT1B\u0026nbsp;receptors compared to\u0026nbsp;sumatriptan underscoring its enhanced receptor specificity and therapeutic potential(7,8).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNaratriptan can penetrate the intact\u0026nbsp;blood-brain\u0026nbsp;barrier (BBB) and exert its pharmacological effects on the trigeminovascular complex, particularly the trigeminocervical complex in the brainstem. However, achieving optimal therapeutic efficacy necessitates a drug delivery system capable of transporting the drug across the BBB in sufficient concentrations. Despite its clinical availability for over two decades, an efficient oral drug delivery system that specifically targets naratriptan to the brain has yet to be developed(9–11).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe BBB, formed by tightly interconnected capillary endothelial cells, represents a significant obstacle in delivering therapeutic agents to the central nervous system (CNS). Effective drug delivery requires either surpassing or transiently disrupting this barrier without compromising its integrity. Innovative strategies, including nanoparticle-based carriers, ligand-mediated transport, or receptor-specific targeting, may offer a potential solution for overcoming these limitations and enhancing the CNS bioavailability of naratriptan, Developing such advanced delivery systems could transform the therapeutic landscape of migraine management by maximizing the efficacy of this potent triptan while minimizing systemic side effects.(12–14).\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;nanostructured\u0026nbsp;lipid\u0026nbsp;carrier’s\u0026nbsp;drug\u0026nbsp;delivery\u0026nbsp;system\u0026nbsp;have developed as a promising drug delivery system for overcoming the blood brain barrier and enhancing therapeutics efficacy in brain targeted treatments.\u0026nbsp;(15). For\u0026nbsp;minimizing\u0026nbsp;the\u0026nbsp;above-mentioned\u0026nbsp;limitation\u0026nbsp;associated with\u0026nbsp;conventional\u0026nbsp;regimen\u0026nbsp;and\u0026nbsp;achieve precise\u0026nbsp;targeting, naratriptan-loaded\u0026nbsp;nanostructured\u0026nbsp;lipid\u0026nbsp;carriers\u0026nbsp;are\u0026nbsp;proposed.\u0026nbsp;The proposed\u0026nbsp;formulation\u0026nbsp;aims to achieve a high local drug concentration at a target site, thereby reducing the required dose and dosing frequency while significantly enhancing patient compliance(16–19).\u0026nbsp;Nanostructured lipid carrier (NLCs), typically composed of a mixture of solid and liquid lipids in ratios ranging from 70:30 to 99:1, offers unique advantages. By blending these lipids in optimized proportion, the system provides increased capacity for effective drug encapsulation mostly\u0026nbsp;the\u0026nbsp;mixture\u0026nbsp;of\u0026nbsp;solid\u0026nbsp;and\u0026nbsp;liquid\u0026nbsp;lipids from\u0026nbsp;70:30 to\u0026nbsp;99:1 offers a unique advantage. By blending\u0026nbsp;these\u0026nbsp;lipids in\u0026nbsp;optimizing proportions, the system provides increasing spital capacity for effective drug encapsulation,\u0026nbsp;in\u0026nbsp;recent\u0026nbsp;years,\u0026nbsp;NLCs\u0026nbsp;have\u0026nbsp;emerged\u0026nbsp;as\u0026nbsp;superior drug delivery platforms due to their exceptional stability, biocompatibility, and the utilization of biodegradable excipients classified as generally regarded as safe. Additionally, their ability to provide a sustained drug release profile further underscores their potential as an advanced carrier system for therapeutic application (18,20,21).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;Preformulation studies are pivotal in providing comprehensive insights into the solubility, stability, permeability, and physicochemical properties of active pharmaceutical ingredients and excipients. These studies form the foundation for the rational design and development of pharmaceutical formulation. A critical component of preformulation involves evaluating the potential interaction between excipients and naratriptan, which can be either antagonistic or synergistic. Favorable interactions are particularly significant as they can enhance the pharmaceutical properties of naratriptan, including its stability, bioavailability, and pharmacokinetics profile. Conversely, negative interaction can compromise formulation performance underscoring the necessity of thorough investigation. The regulatory authorities emphasize stringent requirements for preformulation studies to ensure that the final formulation achieves optimal safety, stability, and maximum therapeutic efficacy. By identifying and optimizing key interactions during the pre-formulation phase, researchers can address potential challenges ensuring the development of robust and effective nanostructure lipid carrier formulations.(22–27).\u003c/p\u003e\n\u003cp\u003eThe concept of Quality by design has evolved intending to impart desired characteristics in the product by constraining the formulation variables and processing parameters.\u003c/p\u003e\n\u003cp\u003eThis study employs a Quality by Design (QbD) approach to optimize, prepare and characterize naratriptan-loaded nanostructure lipid carriers (NPNLC). These advanced nanocarriers have been developed as a brain-targeted delivery system to enhance naratriptan's central nervous system activity while minimizing its systemic exposure in peripheral tissues. This targeted approach amplifies the drug's antimigraine efficacy at lower doses and reduces associated side effects, offering significant advantages over conventional oral dosage forms such as tablets. By leveraging the QbD framework, the study ensures a systematic and robust development process, highlighting the potential of NPNLCs as an innovative and superior therapeutic modality for migraine management. (28–30)\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 \u0026nbsp; \u0026nbsp;Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNaratriptan hydrochloride was generously provided by USV Pharmaceutical Ltd. Mumbai, India \u003cstrong\u003eSolid Lipid Excipients, including Gelucire\u003c/strong\u003e\u003csup\u003e®\u003c/sup\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e50/13 (Stearoyl macrogol-32 \u0026nbsp; glyceride), Geleol\u003csup\u003e®\u003c/sup\u003e Pellets (Glyceryl \u0026nbsp; Palmitostearate), Compritol\u003csup\u003e®\u003c/sup\u003e 888 (Glyceryl behenate), Glyceryl Monostearate (monostearic acid ester of glycerol), and \u0026nbsp; Precirol\u003csup\u003e®\u0026nbsp;\u003c/sup\u003e\u0026nbsp; ATO (Glyceryl stearate), were obtained as gigft sample from Gattefosse, France and Mohini Organic Pvt. Ltd., Mumbai, India.\u003c/p\u003e\n\u003cp\u003eMedium\u0026nbsp;chain\u0026nbsp;triglyceride, including Capryol®\u0026nbsp;90\u0026nbsp;(propylene\u0026nbsp;glycol\u0026nbsp;monocaprylate),\u0026nbsp;Miglyol® 808,\u0026nbsp;Captex®\u0026nbsp;200P,\u0026nbsp;Maisine® 35-1\u0026nbsp;(Glycerol\u0026nbsp;monolinoleate),\u0026nbsp;Campul\u003csup\u003e®\u0026nbsp;\u003c/sup\u003eMCM,\u0026nbsp;and\u0026nbsp;Capryol\u003csup\u003e®\u003c/sup\u003e PGMC (Propylene glycol II monocaprylate were Provided by to ABITEC Mumbai and Mohini Organic Pvt. Ltd. Mumbai as gift Sample. Pluronic\u003csup\u003e®\u003c/sup\u003e F127 was sourced from Sigma-Aldrich and sodium taurocholate from SD fine-Chen Limited, Mumbai. India. \u0026nbsp;High-performance liquid chromatography (HPLC) grade Acetonitrile and water were purchased from SD Fine-chem Limited Mumbai. All other chemicals and reagents used during the study were of suitable analytical grade and were of suitable analytical grade and were used as received.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Solubility Assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe solubility of naratriptan hydrochloride was determined using the water bath shaking method (Remi water bath shaker), Excess amounts of naratriptan were added to 2.0g of various oils, surfactants and co-surfactants. The mixtures were vortexed thoroughly to ensure proper mixing of naratriptan with the excipients and then equilibrated at 37 ±3\u003csup\u003e0\u003c/sup\u003eC in a water bath shaker. After 48 hours of incubation, the mixture were centrifuges at 12,000 rpm for 15 minutes to separate undissolved drug particles. The supernatant was filtered through a 0.45 µm\u0026nbsp;membrane filter, and the filtrate was diluted with methanol. The concentration of naratriptan in the filtrate was analyzed using a UV-Vis spectrophotometer (UV-1800,Shimadzu,Japan)(31,32).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 \u0026nbsp; \u0026nbsp;Compatibility studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompatibility studies were performed to assess potential chemical interaction between naratriptan hydrochloride and the selected solid and liquid lipid material during the formulation of nanostructured lipid carriers (NLCs). Fourier Transform Infrared Spectroscopy (FTIR) was utilized for this purpose. FTIR spectra were recorded using a Bruker Spectrometer with the KBR pellet method over a wavelength range of 400-4000cm\u003csup\u003e-1\u003c/sup\u003e.the scanning speed was set at 2 min\u003csup\u003e-1\u003c/sup\u003e, with a normal slit width ensuring optimal resolution.(33).\u003c/p\u003e\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n"},{"header":"3. Preparation and Experimental Design of naratriptan NLC","content":"\u003cp\u003eNaratriptan nanostructured\u0026nbsp;lipid\u0026nbsp;carrier\u0026nbsp;(NPNLC) were prepared using the melt-emulsification-ultra sonication\u0026nbsp;process. The process includes separate processing of the lipid and aqueous phases. Glyceryl\u0026nbsp;monostearate (solid lipids) and Capmul MCM (liquid lipid) in a 7:3\u0026nbsp;w/w\u0026nbsp;ratio\u0026nbsp;were\u0026nbsp;accurately weighed and heated to 75\u003csup\u003e0\u003c/sup\u003eC which is 5\u0026nbsp;to\u0026nbsp;10\u003csup\u003eo\u003c/sup\u003eC above melting point of the solid lipid, to prevent recrystallization during the process. Naratriptan was dissolved in the melted lipid phase.\u0026nbsp;\u003c/p\u003e\u003cp\u003eSimultaneously, an aqueous surfactant phase was prepared by dissolving Pluronic F 127 (nonionic surfactant) and sodium taurocholate (bile salt) in Millipore water. The surfactant solution was maintained at 75\u003csup\u003e0\u003c/sup\u003eC under continuous stirring using an electronic magnetic stirrer, Mumbai). The aqueous surfactant phase using a borosilicate glass syringe while stirring at 5-10\u003csup\u003e0\u003c/sup\u003eC above melting point of solid lipid\u003c/p\u003e\u003cp\u003eThe resulting coarse emulsion was subjected to probe ultrasonication (probe Ultrasonicator) in a water bath maintained at 750C for 15 minutes at 40 %amplitude. Finally, the prepared NPNLCs were cooled to room temperature on a magnetic stirrer, allowing the lipids to recrystallize and form the nanostructured lipid carriers. (29,34–37).\u0026nbsp;\u003c/p\u003e\u003ch2\u003eLyophilization of NPNLC formulation\u0026nbsp;\u003c/h2\u003e\u003cp\u003eThe NPNLC dispersions were lyophilized to obtain formulation in dry form of the formulation. Initially, the dispersions were frozen at −20ºC overnight, followed by lyophilization at −70ºC for 48 hours. The resulting lyophilized NPNLC powder was carefully collected and stored under appropriate conditions. Subsequently, the formulation underwent comprehensive physicochemical characterization and was subjected to in-vitro, ex-vivo, and in-vivo evaluation to assess its performance and therapeutic potential.(38)\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e3.1 Optimization using Box-Behnken Design.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\u003cp\u003ePreliminary studies were conducted to identify the key factor influencing the particle size, entrapment efficiency, and zeta potential of nanostructured lipid carriers (NLCs). These investigations highlighted the lipid concentration in the formulation as the most critical parameter. A randomized Box-Behnken Design (BBD) was employed to systematically evaluate the influence of three independent factors, each at two levels, on the dependent variables, namely particle size, entrapment efficiency, and percentage yield. The experimental matrix comprised 17 runs, including five center points to assess pure error, ensuring statistical robustness. The NLC formulation was prepared in a randomized sequence to minimize bias and ensure the reliability of the results.(39)\u003c/p\u003e\u003cp\u003eThe experimental design is summarized in Table 1. The excremental design, model fitting analysis, and all the regressions were performed using the Design Expert ® software version (stat-ease Inc., Minneapolis, MN)(40,40–45).\u003c/p\u003e\u003cp\u003eTable 1: Formulation of Naratriptan according to Box-Behnken Design\u003c/p\u003e\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eStd.\u003c/p\u003e\n \u003c/td\u003e\u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eRun\u003c/p\u003e\n \u003c/td\u003e\u003ctd colspan=\"3\" valign=\"top\" style=\"width: 358px;\"\u003e\n \u003cp\u003eFactor\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e1. Glyceryl\u003c/p\u003e\n \u003cp\u003emonostearate (mg)\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e2. Capmul\u003c/p\u003e\n \u003cp\u003eMCM (mg)\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e3. Pluronic\u003c/p\u003e\n \u003cp\u003eF127 (%)\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e225\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e225\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e225\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e225\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e225\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e225\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e225\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e225\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e225\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e"},{"header":"4. Evaluation and characterization of Naratriptan NLCs","content":"\u003ch3\u003e4.1\u0026nbsp;Zeta potential and Particle size analysis Determination\u003c/h3\u003e\u003cp\u003eThe Zeta potential and average particle size of formulated nanostructure lipid carriers (NPNLC) were evaluated in HPLC grade water. Mesurements were performed in triplicate using \u0026nbsp;photon correlation spectroscopy \u0026nbsp;on Zetasizer Nano ZS90 (Malvern Instruments, UK) opening in automatic mode. The analysis were conducted at 25 0 C with a detection angle of 900, ensuring precise and reproducible results.(46,47).\u003c/p\u003e\u003ch3\u003e4.2 \u0026nbsp; \u0026nbsp;% Entrapment efficiency (EE)\u003c/h3\u003e\u003cp\u003eThe formulated nanostructured lipid carriers (NPNLC) were isolated from the dispersion medium by centrifugation at 15,000 rpm for 20 minutes at 40C. The clear supernatant was carefully decanted and analyzed for the free drug content using UV-visible spectrophotometer at the maximum absorbance wavelength (λ max). A placebo NLC (formulated without the drug) was subjected to identical centrifugation conditions (15,000 rpm, 20 minutes at 40 C) and served as the blank. A standard calibration curve for Naratriptan hydrochloride was constructed by plotting absorbance against concentration (x- axis) to check the free drug content.(48,49).\u0026nbsp;\u003c/p\u003e\u003cp\u003eThe percentage of drugs entrapped was calculated by using the following equation:\u003c/p\u003e\u003cp\u003e\u0026nbsp;\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAlYAAABTCAYAAABd90mFAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAADsMAAA7DAcdvqGQAABdXSURBVHhe7d3fixvX2Qfwr9772J7dXIXWGM0GHFqQccdxKZuAC/aoawgJxNU6DbnpxXbU3DTQNdbaOGFjxyNCLpbUqy0pmNJ0tdiQm0pZ2WBDRzWtsykSTUmhK2EC6dXMKuv8Aee9iM5h5mi0q13P/rD3+4HBq2fOjGbOzGgej845SgkhBIiIiIjokf2fHiAiIiKizWFiRURERJQQJlZERERECWFiRURERJQQJlZERERECWFiRURERJQQJlZERERECWFiRURERJQQJlZERERECWFiRURERJQQJlZERERECWFiRURERJQQJlakNJtN5PN5DA0N6bPWVa1Wkc/nkUql9FlET4Rms4nx8XGkUikMDQ1hampKL6IEQaCupVQqhfHxcQRBoBfbVrxGibYHE6sdls1mkUql1p2KxaK+6LrK5bIeWtfKygo6nY4eXtfhw4fx2Wef6eE9o91uo16v6+HEbOZY7nVJ1lm73caJEydw/vx5+L4Px3Fw5coVNJtNvSjQva4zmQxWVlZQKpVw69Yt3Lx5Uy+2rfb6NUq0XZhY7bADBw7A8zwIISCEgOu6ABCJlUolfbF1tdttXL9+XQ+vKZPJ4OjRo3p4IOl0GsPDw3p4z3j//ff1UGI2cyz3unK5jK+++koPb9rNmzfR6XSQyWQwPDyMy5cvQwiBTCajF0W9XsfS0hJ+8IMfAAAmJiawsrKCiYkJvei22uvXKNF2YWK1w44ePYrR0VE9HDExMYGDBw/q4b6CIEAul9PDtEXK5TJmZ2f1cCJ4LDdOfqVNRLQjBO0qrusKAMLzPH2W0mq1RC6XEwAEAJHL5USj0VDzLctS8wAI27aFEEI0Go3IcpZlRZYTofcfRKlUEqZpqnXJv4UQwvd9Nd/zPFEoFNS26tslhIiNie4227Yd2R85zc/PR8rqXNdV22SapqhUKpH569Wj53nCcRxh27bwfV84jqPWJctVKpWe7QofO9d1hWEYAlp96/VTqVTUthYKBbV8v2OZJLlvcjtN04zUrawHwzBi68H3fXV8DcOIPS7hc8U0TeG6rpoXPr6y7uR5GI4NcjxarZbaDzmF30sntz18jMLnied5kXXJqR+9nNz+8LY3Gg1hmqYwTVP4vi9E9zwK149eh+H9RfdclcuuZa1rVAgh5ufnhW3bwnVdMT8/LwzDUGUw4DWqb1t4chwnUpZoL+j/CUE7Yr3ESt44SqWSem1ZljAMI5IU2Lbd8wFomqb6QG61WsI0TWFZVqTMoIlVqVQShmGo7SyVSurDVHS3S67LcRxRqVTUB7jv+8I0zcj2xcXkvsoP50ajIQzDEIZhqDL9OI4jLMsSrVZL+L6vbt6tVkuIAevR8zxVR3IfZCKVy+XUe8mbr37MHMcRjuOo+pbrl6/D9eO6rvA8TyV66x3LJDmOI0zTVHUlt0HeuD3PUwleqVQSvu+r42WapigUCpFl9eNTKBTUsRChcyV805WxcB3qsUGPh+gmAWslVJJcl9wnmSDoic2g14Xocz6Et911XVEqlVSdzM/PR85VuQ1yed/3hWVZ6lz1PE8YhrHuObHeNer7fqT+SqWSKBQKKnHVr8e4mOjWoWVZqg7luaL/p41orxjsk4K2zXqJlUwYwhqNRs/NJe5mHE4kRPeGp98sBrmBtFqt2JuP/ECV5A1GLyf6bJ8ek9sib8jhWL/6EaH6CC8nbyryw34j9aiX07cz7kYqn0qEyZtY+AYJIPYJSXhd+vslLZfLRfZZbqe+Dfp5EXeu6MdHniv600I9gYzb77jYIMdDDJhYzc/PC4QSSBFKHvTkMG5f+4nbbtHdTv2cEN3rMnyu+r4vEDoPS6VSz5Mfee32S14GvUZFt6709Ys+9arH5L7qTzgHqX+iJxXbWD1mZmdncezYsUgsk8nAsiwsLCxE4jrZgLZeryOfz+PKlSt6kYHcvn0bAPD8889H4v0axn7ve9/TQwPZv38/AOB///ufPgvPPPOMHlJqtRrQbawrTUxMINzYeCP1GLdf8j36qdVqaLVakZ6dp0+fBrptgML27dsXeQ0A9+7d00Nbplwuo1wuo91uY2pqCq+//rpeZNNkT7jDhw9H4m+88QYwQD3G2czxiHP9+nWYphlZ3/DwMHK5HDqdzpb08hwZGYm8rtfr6HQ6ME1TnSdPP/00AGBpaQkA8Mknn2B2djZyLslr98svv4ysT9roNXro0CE9NJCnnnoKAGI7CmykXSjRk4SJ1WNoZWVFD/X9wAxrt9vIZrP4zW9+gxdffFH1QNyo1dVVQEtctsKrr74Ky7Lw4YcfIggCBEGAGzduwLbtRN57s/U4KNu2IXt2hqdr167pRR9JsVjsGZ4jbuo3ZEcQBJiamoJlWTh48CD+9Kc/6UUe2bfffht5HZdM7oS4c0Am9NtJP0eEEFheXlbzXdftmS+EwPj4eGQ90nZdo5lMBo7j4Pe//z3a7TYA4M9//jMMw8DJkyf14kR7AhOrx1Cr1dJDAADDMPSQEgQBLMtCOp3GZ5991vcDeSP0Jy9JGx4exkcffYRvvvkGzz77LJ5++mkcO3YMH3/8sV40Vtz2hcc22kw9bkStVosdFLJareqhRzI5Odlzw42bJicn9UUBAL/4xS9w69Yt/Pe//8XExMSWJD1ff/21HgJ2KIkJ63Q6sccIoacx2yHunAjHbty4EZmH7jW93lO1uGsgadPT07AsC6dOnUIqlUK73cbdu3cT/U8K0eOEidVjJpfLYWlpqecDNQiCNZOlL7/8Ep1OB6+99po+a8PkI/7NfP0Spt/Q4l6fOHECi4uLWFlZgeg+7VnvA/snP/kJAODcuXOR+NzcHJ577jngEepxULZtAwAuXrwYicfdQHdarVbDmTNn1q3XzZD1MDMzE4k/fPgQAHqeasi4/vdWeOWVVwAAf/jDHyLx1dVVmKYZGaNKPgEaxEa2e3R0FIZh4NKlS5Hzv91u44svvgC627m0tNQz4OkHH3ygzmfddl2j6Cbmb775JpaXlyGEwOLiYuz4XkR7ht7oinZOuEdNuJF5mOwZJ3sRiVDvn3ADWNk7S/b8kQ2zZSNVz/NUg2TZu0out1ajWEnfTtlYWzba9bvDCYQb4YbJnk+lUklUKhXVc0z2YvJ9XzWCtSxLNZotFArCdd3IvsaR+2Z3eyLKHnrSIPUYbsgcbuAs913GZN263R6PstGyrEuzO7yA0+1uL8n6CTfylQ3Hw0Mu6McyabIe/G5vxfCxKXV7Aer7LPqcK7JRdbgxc3h9ItQDM7yPsrG1ZVnC8zzhuq5aV6FQEI3usA6DHA/R3SdZ1/3qTO5XuOec1+1xF15Gvq++r/3Ieglfw2utQ54HhmGIQqEgCoWCMENDMYSXleez/Hctg1yj8twNv580yDUqxHeN32VvQdu2hRPq5Uq0FzGx2iVkEqFPcRra2E65XK4n0ZCJgxka40feqMzQ2EkI3cTD68Q6vXr8ULdwwzDUh30ulxOVSkX1opJTOKEQoZurnOd3h0RwHEd9IMsbnywXnvSbq07fvvBNXFqvHvX3jDtGclvle4Xfx9fGSHK63fqFNk4TQjdMPSb6HMskyfGLjG6v0VZ3KAp5XOL2WY/J46/HJDc0npdpmrH/cQgnGPPz88LrDlEgkzv9PeO2Qx4P2eNP7kM/4fMEocROinsP/VwO08uiew3rMT3pKIXGm7K7Y12FtUJjrsnrbT36NaBfo3H7FjbINSpCPWz1dcXtJ9FekBLfXfREu06z2cTf//73np8CabfbKBQKePPNN9cdtZ6Itk4QBPjggw9w+fLlnrj8irVf2z6iJxUTKyIiIqKEsPE6ERERUUKYWBERERElhIkVERERUUKYWBERERElhIkVERERUUIST6zK5TJGRkaQSqUwNTWlz1ampqYGGoVa/72zuEn+Blq9Xu+ZFzfpo20TERHtZfJH2Nf65Qn5yxSpVApDQ0OYm5vTiwBaHpDNZtXvSO4ViSZW7XYbZ8+exczMDBqNBq5cuRKbxFSrVezfvx9jY2P6rB5CCPVjwXE/ROp5Hu7cuQN0fx5CCKF+RsPzvJ7yruvi3r17kfcgIiLaq9rtNv7zn/9gYWEB33zzjT4b6CZVx48fx9DQEHzfx927d3H+/PmeH3efm5tDPp/H9evXIYRAOp2GZVmxP4f0pEo0sbp58yYAYGxsDJlMBrZt48MPP4yUabfb+OMf/7ihQePkb7/FGR0dxU9/+tNITH8dtpH3JSIietKl02mMjY1hZGREn6VcvHgRrVYL09PTGB4eRiaTwXvvvYdz586pJ1Ltdhu/+tWv4DiOGrz52rVravm9ItHEKo6e/ebzefzud7+LxB7VRpOljZYnIiLarbLZbE9zl2Kx2NNc5lGUy2XYth35sfYf//jHQOihyu3btwEAP/vZz1QZADh16hRmZ2cjsSfZlidWBw4cUH/n83lcuHAhcmAeRbvd7vsdb5xqtRr71WSY3h6r30RERLQbLC4uqiYzuVwOo6OjmJychGma8DzvkR8m1Ot1dDod/OhHP4rEM5kMAKjmOJ988gkA4LnnnouUO3r0KNBdz16QaGL16quvAt3KazabqNVqePnll4FutpvJZDA6OopmszlQA3fduXPnIsmNaZpYXV3ViykvvPBCpPzp06f1Ij30Nln9piS9/fbbPYkbJ06cOHHafVOj0dA/wneFyclJOI6DhYUFVKtV1c4pyd9T3b9/vx6KldTDk8dVoolVOp3G/Pw8XnrpJRw5cgSFQgHj4+NoNpv461//iomJCQRBgBMnTuC3v/0tfN/HwsLCwI8p9cbrnufpRSL0xusyo99t3n777Z7EjRMnTpw47b7pyJEj+kf4rjE9PQ3LsnD69Gn1IIO2X6KJFQCMj49jZWUFQghcvnwZQRDgvffew/T0NADgH//4BzqdDk6ePInh4WGcOnUKN27c0FczkNHRURw8eFAP9yWfqK1F/99Jv4mIiGg3GR4exjvvvAOEvpaj7Zd4YqW7ePEizp8/rx4NfvHFF0D36RYAHDp0CEtLS5FlNmKtMTd06XR63Qxe/99Jv4mIiGg3CYIAMzMzKJVKqNVqA38btJ7R0VEYhqHaUkmyzZTsif/KK69E4lJ4SKS9YEsTq7m5Obz44ouqgRsA/PCHPwS6Dc8B4MGDB7AsS83fjK1oxE5ERPS4CIIAv/71r/Hxxx9jYmIChUIB586dG2gg7kGMj4/j/v37kfGo5JiQ8tugkydPRuLobtf9+/fhOI6KPem2LLFqNptoNps9T5SOHz8OwzBw+/ZtBEGAcrmMM2fORMroPv30UwCIbaher9eRy+XUAQ2CQGXHDx8+1Ep/14j+0qVLeyZzJiKiJ1sQBMhms3j55ZfVt0NvvfUWAOD1118f6EFCEAQqCYobzFM255HjUdXrdVy9ehWu66pvoNLpNFzXxdWrV9FsNhEEgSovl98TxBbwfV/kcjnh+74+SwghRKPREKZpCgCiUCjosyMArDvZti2EEMLzvJ55cZPruvrbEBERPZZs21b3t7gYAOF5XmSZsLh7Z9x9stFoqPWapilKpZJeRAghhOu6wjAMAUDkcjnRarX0Ik+0lGCDISIiIqJEbNlXgURERER7DRMrIiIiooQwsSIiIiJKCBMrIiIiooQwsSIiIiJKCBMrIiIiooQwsSIiIiJKCBMrIiIiooQwsaI9Rf6M0rFjxzb8A6XyNylHRkY2vCwREe0NTKxoy9XrdaRSqYGmjWq32wP9DlbYvn37sLS0pIcH8v3vfx+tVksP7xnlclkPJWYzx5KIaLdhYkXbwnEc+L4PIQTkryjZtq1et1otWJalL7au999/Xw+taXh4GGNjY3p4IOl0Gvv27dPDe0a73cb169f1cGI2eiyJiHYjJla05e7du4dr166pX12Pk06n8c4778T+qno/5XIZs7Ozepi2QBAEyOVyejgxPJZE9KRgYkVbbnJyUg/FGhsbiyRfsj1TKpXqaddUrVZx9uxZAMALL7yAVCqlvkYqFotquaGhIUxNTanlNqrdbmN8fFyt6913343Mr9fryOfzyGazaDabGBkZwcjICI4dO6a+3gxvlx6TisUihoaGer4aHRkZiZTTNZtNtX2pVAr5fL4nOV2rHoMgUPPr9Tqq1aoqG663bDaLpaUl1Go1pFIpZLNZNa/ZbCKbzcbWd7h+giBAPp9X29FsNoF1jiUR0WNHEO0AAMK2bT2sFAoFYVmWaLVaQgghSqWSACAcx1FlPM8TAITneSo2Pz8fibmuKwCISqWiyoju+7uuG4npfN8XpmkKx3GE7/ui1WoJy7Iiy3qeJ0zTFJZlCdd1RalUUtsttzm8fXGxQqEgDMMQjUZDCCGE4zgCgCiVSqpMnEajIQzDUOUqlUpPHa1Xj61WS9WR4zjCdV3heZ7I5XICgNomIYSwbbvnmDUaDWGaptofuf64+nEcR1QqFbWduVxOrSfuWBIRPY6YWNGOwBqJVavVik2G9Jt93M24VCoJwzDUa9/3Y5OouJjOcRxhmmYkJpOC8LK2bfeUE322Ly6mJ0My1q9+pFwu17OcYRgqYdloPYbLxW1nXGKVy+XE/Px8JGZZVuQY2LYtLMuKlNHXFfd+RESPI34VSLvOzZs3AQCHDx+OxN944w0AQK1Wi8TDJiYmsLKyor7iOn78uF5kYOVyGadOnYrE+jVeX+8ru7UYhoF2u62HkU6n9VDEwsICDh06FImtrKyonnsbrce4fbt3754eilhYWMDZs2cjX18uLS2h0+lE9imufZ3+/kRETwImVrRrffvtt5HXcTf+OHNzc3j22Wfx1Vdf4datW/rsgXU6nZ7EZStcu3YNtVpNtSuSidFrr72mldyczdbjoDzPU707w9N6iSER0ZOIiRXtWl9//bUeAgDs379fDynFYhHnz5/H3bt3cfny5Ue+uf/zn//UQ4kbHx+H4zh49913kUqlcOHCBVQqFYyOjupFe8Rtnz7W1GbqcSM+/fRTPYRqtaqHiIj2BCZWtOvYtg0AmJmZicQfPnwIADh58mQkHnbnzh08//zzyGQy+qwNM00Tt27d6ullt1Fyu/W/pWKxiEOHDmFxcRFCCCwvLw801pZt21hYWIj0oAuCAP/617/UfGyyHgeVy+UwOzurevhJf/nLXyKviYj2CiZWtO3kE5Xl5eXYpCWTycBxHNRqNczNzQHdYQ8uXbqEQqGgnkI99dRTQLcdUBAEqNfrOHDgAO7fv49ms6naWQHAgwcP1DADMgmIe9oTNjMzg06ng2w2q9oLyaczDx48UO+xvLyMWq3Wk1w888wzAIBLly6hXq+jWCzib3/7m1qPLH/nzh1cvXoV2WwW2WwW4+PjKBaLPU+edBcuXAAAvPTSS5iamkKxWEQ2m8XPf/5zYAP1+O9//xvQ2lPJ5Gt1dVXFZN0GQaCeSJ0/fx6dTgdHjhxBPp9XQ13IrzFl/cjlJPm3/DfuWBIRPZb01uxEW8m2bQEgMvXrnee6rjAMQwAQpmnGDj8ghyYoFApChLr/y5jv+8KyLGGapmg0Gqr3WXhaS6VSUeuzbVuUSiVhmqZwXVf1ugtPeq82OfyAYRhifn5eDT9QKpWE7/tCdIeIME1T7Wt46lc3Unj7LMuKDI8grVWPcqgFOdm2HRsToeEdZF1KnuepYShM04z0LtT3J67+ZZ3px5KI6HGUEt99+BHRDpmamsJbb73V03OuWq1iZmYGi4uLkTgREe1e/CqQaAcVi0V8/vnnPUkVABw/fvyRG98TEdH2YmJFtINWV1dRq9UwNTWFer2OIAjQbDZRrVZx8eJFTE9P64sQEdEuxq8CiXbY3NwcPvroIywtLQEALMvCmTNn8Mtf/jL2SRYREe1eTKyIiIiIEsKvAomIiIgSwsSKiIiIKCFMrIiIiIgSwsSKiIiIKCFMrIiIiIgSwsSKiIiIKCFMrIiIiIgSwsSKiIiIKCFMrIiIiIgSwsSKiIiIKCFMrIiIiIgS8v9dbr44yhf1aQAAAABJRU5ErkJggg==\"\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e4.3 In vitro Drug studies:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe in vitro drug release profile of all the formulated NPNLC batches was determined using a USP Type II dissolution apparatus (TDT-08L Electrolab, India). Drug-loaded NLCs, equivalent to 5mg of Naratriptan hydrochloride,were redispersed in 2 mL of \u0026nbsp;phosphate buffer (pH 6.4) and enclosed in a pre-soaked dialysis membrane bag. The dialysis bag was securely tied to the paddle of the dissolution apparatus and immersed in 500 ml phosphate buffer (pH 6.4), serving as the dissolution medium. Prior to experimentation, the dialysis membrane was soaked in deionized water for one hour to ensure optimal permeability. The dissolution medium was maintained at a temperature of 37°C ± 1°C and agitated at a speed of 100 rpm respectively. At predefined time intervals, 5 mL aliquots were withdrawn and immediately replace with an equal volume of fresh phosphate buffer (pH 6.4) to maintain the sink conditions. The percentage cumulative drug release was estimated by measuring the UV absorbance of the aliquots, at λmax = 226 nm, against the blank dissolution medium as the reference. All experiments were performed in triplicate, and results are presented as mean % drug release ± standard deviation. The drug release mechanism was elucidated by fitting the in vitro release data to various release kinetic models, including zero order, first order, Higuchi model, Hixson-Crowell, and Korseymere Peppas models, using appropriate mathematical software. \u0026nbsp;(41,42) .\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e4.4 \u0026nbsp;Fourier- Transform Infrared (FTIR) Spectroscopy\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe FTIR spectra of lyophilized optimized drug-loaded NPNLC batch, the physical mixture of Naratriptan hydrochloride and excipients in proportions used for fabricating NPNLC, Placebo nanoparticles (formulated without drug ), and pure Naratriptan hydrochloride were recorded using an FTIR spectrophotometer (Perkin Elmer, USA) in the frequency range of 400–4000 cm−1. The KBr pellet method was employed to observe the potential chemical interactions between the pure drug and excipients within the formulation. (50,51) .\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e4.5 Powder wide-angle X-ray diffraction\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe XRD patterns of pure Naratriptan hydrochloride and optimized NPNLC formulation were analyzed using an X-ray Diffractometer (Rigaker Geiger flex, Japan). Prior to analysis, the NPNLC formulation was lyophilized to ensure accurate characterization. Diffractograms were recorded from the initial angle 2θ =100 to the final angle of 400 with a CuKα radiation source. Samples were mounted in the glass sample holders and scanned over a temperature range of 20°C to 80°C with a scan rate 2o/min, under operating conditions of \u0026nbsp;35kV and 40 mA current. (52,53) .\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e4.6 Differential Scanning Calorimetry (DSC):\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eDifferential Scanning Calorimetry (DSC) thermal analysis was performed to evaluate the physical state and thermal behavior of the pure Naratriptan Hydrochloride, and the optimized NPNLC formulation. Thermogram were recorded on DSC analyzer (TA Instruments DSC SDT Q600), to determine the physical nature of the drug and the polymers/carriers in the formulation. Approximately 4–5 mg of each sample was accurately weighed, sealed in aluminum pans, and hermetically crimped to prevent moisture loss and ensure uniform heat distribution. The samples were scanned over a temperature range of 25–400◦C at a heating flow rate of 10◦C/min under a continuous nitrogen purged maintained at a flow rate of 100 mL/min. The data was analyzed using the Universal Analysis 2000 software package (SDT Instruments). (40,54–57) .\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e4.7 Scanning electron microscopy (SEM)\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe surface morphology study was carried out to examine the surface morphology and shape (Jeol Japan 6000). The surface morphology of the optimized NPNLC formulation were analyzed using a Scanning Electron microscope (SEM, JEOL JSM-6000 Japan). SEM employs a focused beam of electrons, rather than light, to generate highly magnified images by interacting with the sample surface, leading to the emission of secondary electrons and X-rays. Prior to imaging, the samples were carefully mounted onto metal stubs using a double-sided adhesive tape and sputter coated with a thin conductive layer under a vacuum. The analysis was performed at various magnifications, at an acceleration voltage of 15 kV, and with a working distance maintained at 20 μm. Microphotographs were taken at different magnifications, and surface morphology was examined at a higher magnification. (50,52) .\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e4.8 Ex vivo drug release study\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eAn ex vivo drug diffusion study was conducted using freshly excised sheep nasal tissue to evaluate the drug release characteristics of Naratriptan hydrochloride and its nanostructed lipid carrier (NPNLC) formulation. Freshly excised sheep nasal tissue was collected from a local slaughterhouse, immediately immersed in freshly prepared phosphate buffer solution (pH 6.4), and transported to the laboratory. The bony cartilage was carefully removed from the mucosal membrane; and the nasal tissue was thoroughly washed and stabilized in a phosphate buffer (pH 6.4) before experimentation. \u0026nbsp;The tissue was mounted between the donor and receptor compartments of a Franz diffusion cell under a phosphate buffer solution (pH 6.4). In the donor compartment 1mL of Naratriptan Hydrochloride pure drug solution (5 mg/mL) was introduced onto the nasal tissue, while the receptor compartment was filled with 50 mL of phosphate buffer solution (pH 6.4).\u0026nbsp;\u003c/p\u003e\u003cp\u003eThe stirring speed was maintained at 200 rpm using a magnetic stirrer, and the temperature was kept constant at 37±50C to mimic physiological conditions. For NPNLC formulation, lyophilized NPNLCequivalent amount to 5 mg of Naratriptan was reconstituted in 1 mL of phosphate buffer solution (pH 6.4) and applied to the nasal tissue in the donor compartment. Aliquot 2mL of Samples at predetermined time intervals (0.5, 1, 2, 4, 6, 8, 10, 12, and 24 hours) were withdrawn from receptor compartment and immediately replaced with an equal volume of fresh phosphate buffer (pH 6.4) to maintain sink conditions. The amount of drug diffused across the nasal tissue was analyzed using a UV–visible spectrophotometer (Wenser) at a wavelength λmax 226 nm. The percentage of \u0026nbsp;drug diffusion was calculated using a pre-established standard calibration curve for Naratriptan hydrochloride. (55–59) .\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e4.9 \u0026nbsp;Stability Investigation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u0026nbsp;The stability of the lyophilized NPNLC formulation was evaluated by storing the samples in a screw-capped glass bottles covered with aluminum foil to protect them from light exposure. The samples were maintained under two storage conditions: refrigerated temperature (4-8o C) and a room temperature (250C) for a period of 1-6 months. At predetermined intervals, the formulations were analyzed for percent encapsulation efficiency (% EE) and Drug content. The results were \u0026nbsp;compared with a freshly prepared NPNLC formulations. (50,60) .\u0026nbsp;\u003c/p\u003e"},{"header":"5. In vivo Studies","content":"\u003cp\u003eAdult male Swiss albino mice (25-30 g) and adult male Wistar rats (200-250 g) were utilized in the present study. The animal Activity protocols for the optimized formulation were approved by Dr. D. Y. Patil Institute of Pharmaceutical Sciences \u0026amp; Research, Sant Tukaram Nagar, Pimpri, Pune 411018 with Institutional Animal \u0026nbsp; Ethical \u0026nbsp; \u0026nbsp;Approval \u0026nbsp; No \u0026nbsp; DYPIPSR/IAEC/OCT/21-22P-19, \u0026nbsp; in compliance with the guidelines established by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. Prior to experimentation, animals were acclimatization to the laboratory conditions for at least one week. They were housed under a controlled environment with \u0026nbsp; a 12 hour light/dark cycle, at a temperature of 25\u0026deg; \u0026plusmn; 1\u0026deg;C. Standard laboratory diet and water were provided ad libitum throughout the study period.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.1 Pharmacodynamic study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe optimized nanostructured lipid carrier (NLC) formulation was evaluated for its efficacy in migraine management using established behavioral models. The acetic acid-induced writhing test was employed to assess hyperalgesia, while the light /dark box test was utilized to evaluate photophobia, two hallmark symptoms of migraine. These studies were conducted Swiss albino mice, ensuring relevance to migraine symptomatology and their therapeutic potential. (60) .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.1.1 Acetic acid-induced writhing test:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdult male Swiss albino mice were randomly assigned to four different groups (n=6 per group) :control (normal saline), standard drug (NP), placebo formulation (NLC), and the optimized test formulation (NPNLC). The drug and test formulations were orally administered after dispersion in deionized water. Hyperalgesia was induced by intraperitoneal injection of \u0026nbsp;Acetic acid solution (0.3%, 10 mL/kg). Five minutes after acetic acid administration, the number of abdominal writhes was counted over a 10 minute observation period.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.1.2 Light/dark box model:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhotophobia, a hallmark symptoms of migraine, was evaluated in Swiss male albino mice in terms of light aversiveness using light and dark box models (61) .\u003c/p\u003e\n\u003cp\u003eThe light/ dark box apparatus consisted of a rectangular box (60x60x45 cm3), divided equally into two compartments connected by an opening (7x7 cm2). The light compartment (LC) was illuminated by a 60w bulb and left uncovered from the top, while the dark compartment was completely painted black and fully enclosed on all sides.\u003c/p\u003e\n\u003cp\u003eMice were assigned to treatment groups, including NPNLC, Placebo (NLC), and control and were administered their respective treatments for seven consecutive days. On the experimental day, each of the animals was placed in a light compartment and observed for 5 minutes. The time spent in the light compartment was recorded and expressed as a percentage of the total observation period, calculated as follows:\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.1.3 \u0026nbsp; Determination of Brain Uptake Potential\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo of the output responses for formulation optimization were drug concentration in the brain and the AUC brain/AUC plasma 2 hours after drug administration. Male Wister rats were divided into 4 groups (n=3): normal saline (control), std. (5 mg/kg by nasal route NP), placebo (NLC), and formulated formulation (5 mg/kg by nasal route NPNLC). Prior to the trial, animals were fasted overnight with no access to water. The medicine, placebo, control, and test formulation were administered through the nasal route to the complete in vivo trial after 0.5ml blood was administered in heparinized tubes at different times of 30, 75, 135, 225, and 240 minutes. Samples were centrifuged at 8000 rpm for 10 minutes, and the plasma were separated and stored at 20\u0026deg;C till the next processing and analysis. Following blood samples collection, the animals were sacrificed by decapitation under deep anesthesia at various intervals of 30, 75, 135, 225, and 240 minutes. The brain was quickly extracted, weighed, and blotted to remove excess surface blood. The brain tissue was homogenized in 0.01 M phosphate buffer (pH 6.4), then centrifuged at 8000 rpm for 10 minutes. The supernatant was stored at - 200C fo subsequent processing and analysis. In the present study, chromatographic separation was performed using an HPLC system (prominence version 1.24 Sp1, Shimadzu Corporation, Japan), controlled by LC solution software. Before HPLC analysis, plasma and brain samples were subjected to liquid-liquid extraction. a 200\u0026micro;l aliquot of plasma or brain sample was treated with 20\u0026micro;l of standard solution (100 \u0026micro;l/ml and 200\u0026micro;l/ml) and 2M sodium hydroxide (200 \u0026micro;L). After vortexes mixing for 1 minute, 2 ml of ethyl acetate was added, followed by shaking for 2 minutes, and centrifuged at 10000 rpm for 15 min. at 100C. the extraction procedure was repeated twice. The supernatant (organic phase) was collected, evaporated to dryness, and reconstituted with 200 \u0026micro;l mobile phase. The drug was quantified using a validated HPLC method applied to plasma and brain extracts. Prior to injection into HPLC analysis, sample (20 \u0026mu;l) were filtered through a 0.22 \u0026micro;m syringe filter. HPLC analysis was conducted using a Shimadzu Corporation system equipped with Photodiode array (PD) detector and symmetry\u0026reg; C18 column (5\u0026micro;m particle size, 4.6 mm \u0026times; 250 mm). The mobile phase consisted of a mixture of acetonitrile and 0.05 M potassium dihydrogen phosphate (80:20, v/v), with 10% acetonitrile and 0.1 % triethylamine, pH adjusted to 3.8 with o-phosphoric acid. The flow rate was set at 1 ml/min, the total run time was 12 minutes, and UV detection was performed at \u0026lambda; max 226 nm. Brain targeting potential was assessed by determining the drug concentration in both brain and plasma. The ratio of AUC (brain) /AUC (plasma) was calculated after 2 hours of drug administration using the trapezoidal method. The pharmacokinetic parameter was estimated using PK solvers add-in program for the Microsoft Excel. Key parameters, such as maximum plasma concentration (Cmax) and the time to reach maximum plasma concentration (T max) were directly obtained from the plasma concentration versus time curve. The area under the curve (AUC) was calculated for further analysis.\u0026nbsp;\u003cbr\u003eRelative bioavailability was calculated by\u003cbr\u003e\u0026nbsp;(F) = AUC test/AUC reference x100.\u003c/p\u003e"},{"header":"6. Statistical analysis","content":"\u003cp\u003eStatistical analysis was conducted using GraphPad Instata software (version 3.06). Results are presented in terms of mean ± standard deviation (SD) of the mean. One-way analysis of variance (ANOVA) was employed to correlate the statistical significance of the results, followed by the post hoc Tukey-Kramer test to determine the significant differences between groups. The statistical significance was set at a P-value less than 0.05.\u003c/p\u003e"},{"header":"7. Result and discussion","content":"\u003cp\u003eIn this study, the randomized Box-Behnken Design, a quadratic randomized response surface methodology, was used to optimize the formulation for the optimization of formulation for seventeen experimental runs, ensuring a systematic and robust approach to parameter evaluation. The effects of key factor including the amount of solid lipid (Glyceryl monostearate) (A), liquid lipid (Capmul MCM) (B), and surfactant concentrations (C), on critical quality attributes (CQAs) of the prepared NLCs were systematically evaluated. The CQAs assessed namely, percentage yield (R1), entrapment efficiency (% EE) (R2) and particle size (R3). The mean particle size across all the formulation batches ranged from 79.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37 nm to 185.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43 nm, the percentage yield varied between 60.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74% to 75.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15%, while the entrapment efficiency of the formulation ranged from 60.12\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1% to 75.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55%.\u003c/p\u003e\n\u003cp\u003eThe responses were analyzed using the general polynomial equations, and Analysis of Variance (ANOVA) was performed utilizing Design Expert software. Statistical parameters, including degrees of freedom (df), sum of squares, mean sum of squares, and Fischer\u0026rsquo;s value (F-value) were evaluated to value model performance. The statistical significance of the model was determined based on the F-value, P-value, correlation coefficient (R\u003csup\u003e2\u003c/sup\u003e), and adjusted correlation coefficient (adj R\u003csup\u003e2\u003c/sup\u003e). The regression equations derived for the response variables are presented as follows: (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\n\u003cp\u003eThe regression-coded equations derived for the response variables are as follows:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinal Equation in Terms of Coded Factors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePercentage Yield (X\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e)\u0026thinsp;=\u0026thinsp;+\u003c/strong\u003e\u0026thinsp;64.39\u0026thinsp;+\u0026thinsp;3.57 A\u0026thinsp;+\u0026thinsp;2.34 B\u0026thinsp;+\u0026thinsp;4.52 AB\u0026thinsp;+\u0026thinsp;3.53 A\u0026sup2; (\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e\n\u003cp\u003eEntrapment Efficiency (X\u003csub\u003e2\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;+\u0026thinsp;62.25\u0026thinsp;+\u0026thinsp;3.13A\u0026thinsp;+\u0026thinsp;3.7B\u0026thinsp;+\u0026thinsp;4.66A\u0026sup2; (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\n\u003cp\u003eParticle Size (X\u003csub\u003e3\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;+\u0026thinsp;145.32\u0026ndash;31.87 A -15.96 B -5.06 C -18.84 AB -13.71 AC -5.93 BC -9.82 A\u0026sup2; +12.88 B\u0026sup2; -11.15 C\u0026sup2; (\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e\n\u003cp\u003eWhere,\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u0026thinsp;=\u0026thinsp;Conc. of Glyceryl Monostearate\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e\u0026thinsp;=\u0026thinsp;Conc. of Capmul MCM\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e\u0026thinsp;=\u0026thinsp;Pluronic F127\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eX1\u003c/strong\u003e\u0026thinsp;=\u0026thinsp;Percentage Yield\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eX2\u003c/strong\u003e\u0026thinsp;=\u0026thinsp;Entrapment Efficiency\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eX3\u003c/strong\u003e\u0026thinsp;=\u0026thinsp;Particle size\u003c/p\u003e\n\u003cp\u003eFrom Eq.\u0026nbsp;(1) it can be conferred that an increase in concentration of Glyceryl monostreate (A) positively influences the percentage yield (X\u003csub\u003e1\u003c/sub\u003e) and an increase in the concentration of both Glyceryl monostearate (A) and Capmul MCM (B) lead to an increase in the percentage yield. In contrast, the interaction between Glyceryl monostearate (A) and the Pluronic F127 (C) resulted in a reduction in the percentage yield of formulations. From Eq. (2) it is evident that the entrapment efficiency of a formulation increases with the concentration of all three parameters (Glyceryl Monostearate (A), Capmul MCM (B), and Pluronic F127 (C)). However, the interaction between Glyceryl monostearate (A) in combination with the Pluronic F127 (C) leads to decreases the entrapment efficiency when the concentrations of both are decreased. Eq. (3) revels that the particle size of NPNLC decreases with a reduction in the concentration of the Glyceryl monostearate (A), Capmul MCM (B), and Pluronic F127 (C). Apart from this if we double the concentration of Capmul MCM the particle size increases. (Fig. 1)\u003c/p\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eRegression Analysis\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eRegression Analysis\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eResponse\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eThe regression equation for coded factors\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAdjusted R\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAdeq.\u0026nbsp;Precision\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Yield\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eYield\u0026thinsp;=\u0026thinsp;+\u003c/strong\u003e\u0026thinsp;64.39\u0026thinsp;+\u0026thinsp;3.57 A\u0026thinsp;+\u0026thinsp;2.34 B\u0026thinsp;+\u0026thinsp;4.52 AB\u0026thinsp;+\u0026thinsp;3.53 A\u0026sup2;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.8371\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.6277\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.2089\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eAdequate precision value, which measures the signal-to-noise ratio, indicates the reliability of model in predicting responses. A ratio greater than 4 is considered desirable, and your ratio of 7.2089 confirming that the model is suitable for navigating the design space effectively.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e%EE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEntrapment Efficiency\u0026thinsp;=\u0026thinsp;+\u0026thinsp;62.25\u0026thinsp;+\u0026thinsp;3.13 A\u0026thinsp;+\u0026thinsp;3.77 B\u0026thinsp;+\u0026thinsp;4.66 A\u0026sup2;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.8721\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.7076\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e8.8379\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eAdequate Precision value, measures the signal-to-noise ratio. A ratio greater than 4 is considered desirable. Obtained ratio of 8.838 suggests a robust signal, indicating that the model is suitable for navigating and optimizing the design space.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eParticle size\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eParticle Size\u0026thinsp;=\u0026thinsp;+\u0026thinsp;145.32\u0026ndash;31.87 A -15.96 B -5.06 C -18.84 AB -13.71 AC -5.93 BC -9.82 A\u0026sup2; +12.88 B\u0026sup2; -11.15 C\u0026sup2;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.9882\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.9729\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e28.88\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eAdequate Precision, which measures the signal-to-noise ratio, serves as an indicator of the models reliability n predicting responses. A ratio greater than 4 is acceptable, and ratio of 28.885 indicates an adequate signal. This model can be used to effectively navigating and optimizing the design space.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\n \u003ch2\u003e7.2 Lipid Screening\u003c/h2\u003e\n \u003cp\u003eThe various solid and liquid lipids are used for the screening of lipids in NLC production based on the solubility of a drug in lipid selection of lipids are done. In this study, we found that the drug naratriptan showed a higher amount of solubilization in Glyceryl monostearate and Campul MCM. As more solubility of a drug in these lipids, we selected this lipid for feature development of NLC. The selection of surfactant was done based on the Hydrophilic lipophilic balance (HLB scale) and the physicochemical property which is most suitable for the nose-to-brain drug delivery system. (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\n \u003ch2\u003e7.3 Physicochemical Characterization of Naratriptan NLC\u003c/h2\u003e\n \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n \u003ch2\u003e7.3.1 Desirability\u003c/h2\u003e\n \u003cp\u003eThe primary objective of pharmaceutical formulation optimization is to discover the stages of the variable that most significantly influence the selected responses and to determine the optimal levels of these variables from which to develop of robust product with superior-quality attributes. In this study, all the measured responses affecting product quality were considered during the optimization process. The optimization criteria were set to maximize both percentage yield and entrapment efficiency (EE), while minimizing particle size.\u003c/p\u003e\n \u003cp\u003eThe graph shows that the ideal suitability of the NLC composition was 0.983 which is closely related to the ideal value, indicating a strong alignment between the predicted parameters and desired parameter values. (Fig. 3) The predicted formulation shows a composition identical to that of NLCF 2, further validating the accuracy of the optimization model. (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e),\u003c/p\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCharacteristics of optimum formulation\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSN\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eObjects\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGlyceryl monostearate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCapmul MCM\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePluronic F127\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e%\u003c/p\u003e\n \u003cp\u003eYield\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e%EE\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParticle size (nm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDesirability\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePredicted\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.983\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eActual\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e\n \u003ch2\u003e7.3.2 Fourier -Transmission Infrared (FTIR) Spectroscopy\u003c/h2\u003e\n \u003cp\u003eThe FTIR spectrum of pure drug NP exhibited a distinct peak at 2918.93 cm\u003csup\u003e-1\u003c/sup\u003e, corresponding to the CH stretching vibration, a band at 1146.74 cm\u003csup\u003e-1\u003c/sup\u003e attributed to C\u0026thinsp;=\u0026thinsp;O stretching, and a peak at 1064.57 cm\u003csup\u003e-1\u003c/sup\u003e corresponding to S\u0026thinsp;=\u0026thinsp;O stretching vibration band. (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e), The FTIR spectrum of NP formulation represented all the characteristic peaks of both the drug and the excipients, thereby confirming the absence of any chemical interaction between them. (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e,\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eInterpretation of FTIR Graph\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSN\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eName of Functional Group\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWave number cm-1\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC-H Stretching asymmetric\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2918.93\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC-H stretching symmetric\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2851.52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u0026thinsp;=\u0026thinsp;O Stretching\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1146.74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC-N stretching\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1646.74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-S\u0026thinsp;=\u0026thinsp;O stretching\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1064.57\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e\n \u003ch2\u003e7.3.3 Differential Scanning Calorimetry (DSC)\u003c/h2\u003e\n \u003cp\u003eThe DSC thermogram presented in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e reveals critical insights into the melting point and the crystalline or amorphous nature of naratriptan hydrochloride and its lipid carriers. A sharp endothermic peak observed at 226-236\u003csup\u003e0\u003c/sup\u003eC in the thermogram of pure naratriptan hydrochloride indicates its crystalline structure and characteristic melting point. This sharp endothermic peak of naratriptan is absent in the thermogram of naratriptan loaded nanostructured lipid carriers (NPNLCs), indicating that either naratriptan is completely solubilized in the lipid matrix or has transitioned into an amorphous form upon incorporation into the carrier system. Furthermore, the DSC profile of NLCs was investigated and analysis with Glyceryl monostearate and portion amount Campul MCM in the ratio of 70:30 used for the NLCs formulation, indicative of reduced crystallinity of Glyceryl monostearate which shows broadening of the thermal peak. This change in thermal behavior highlights the effective encapsulation of naratriptan within the lipid carriers. (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec30\" class=\"Section3\"\u003e\n \u003ch2\u003e\u003cstrong\u003e7.3.4 Percentage yield (% yield), % Entrapment Efficiency (%EE), and Particle size\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003ePercentage yield (% yield), % Entrapment efficiency (% EE), and Particle size are shown in Table \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eValidation of predicted and experimental values of NLC formulation\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSN\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eResponse\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eExperimental Value\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePredicted Value\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e% Relative Error\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Yield\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e%EE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.94\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eParticle size(nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec31\" class=\"Section3\"\u003e\n \u003ch2\u003e\u003cstrong\u003e7.3.5 Result of Particle Size of NLCF2\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eThe average particle size of NTP-NLC was measured using dynamic light scattering (DLS), Malvern zeta sizer (Malvern zeta sizer, Worcestershire, UK) from SAIF RGPV, Bhopal. The particle size of optimized formulations is as follows for NLCF2 is 79.74. (Fig. 7) The figure indicates the intensity of size distribution here the contributions in terms of % intensity are shown as a function of Particle diameter the Z value was recorded as 79.74 with a width of 17.45.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec32\" class=\"Section3\"\u003e\n \u003ch2\u003e7.3.6 X-ray diffraction study\u003c/h2\u003e\n \u003cp\u003eThe X-ray diffractogram (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e) shows a very high intensity, depicting the crystalline nature of pure naratriptan hydrochloride (NP). In contrast, the X-ray Diffractogram of lyophilized formulation NPNLC shows a significant reduction in peak intensity.This decrease suggests successful incorporation of drug into the lipid carrier system, accompanied by a slight reduction in its degree of crystallinity. These results show that, potentially enhancing its solubility and bioavailability.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e\n \u003ch2\u003e7.3.7 Scanning electron microscope (SEM)\u003c/h2\u003e\n \u003cp\u003eThe optimized naratriptan formulations\u0026apos; spherical shape was confirmed by an SEM photomicrograph. The particles had moderate uniformity and were all discrete entities with no aggregation. They were racially homogenous to smooth surfaces and had no rupture and the size was discovered to be within the limit. (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e)\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e\n \u003ch2\u003e7.3.8 \u003cem\u003eIn vitro\u003c/em\u003e Drug release\u003c/h2\u003e\n \u003cp\u003eThe in vitro drug release profile of naratriptan from NLC formulation was evaluated using the dialysis bag method and compared with a Naratriptan drug solution. The study was conducted in a phosphate buffer (pH 6.4) due to partial solubility of drug in the buffer solution, as reported in literature. The results demonstrated that64.36% of the drug was released from the pure naratriptan solution within 2 hours, whereas the NPNLCs formulation exhibited a sustained release pattern with a cumulative drug release of 99.12% over 24 hours Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e. It was concluded that the released rate from nanostructured lipid carrier could be significantly prolonged the release of Naratriptan. Furthermore, it was observed that the drug release rate decreased with an increase in NLC particle size. The in vitro release data were fitted to various kinetic models, such as zero order, first order, and Higuchi, Korseymere Peppas, and the Hixson-Crowell models, with correlation coefficients (R\u003csup\u003e2\u003c/sup\u003e) of 0.9708,0.9728, 0.9901, 0.9850 and 0.9711 respectively, indicating that the release mechanism is best described by Higuchi model.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec35\" class=\"Section3\"\u003e\n \u003ch2\u003e7.3.9 Ex-vivo diffusion studies\u003c/h2\u003e\n \u003cp\u003eEx-vivo diffusion tests on sheep nasal mucosa for NP solution and NPNLC preparation were carried out. After 24 hours at pH 6.4, the drug\u0026apos;s ex-vivo release for NPNLC was found to be 90.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21%. Whereas, the NP solution demonstrates 76.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56% drug release; it persists in protonated form at nasal mucus layer pH (5.5\u0026ndash;6.5) and is transferred via tight junctions started by sodium taurocholate. Increase permeation through nasal mucosa as a result of paracellular transport across tight junctions. (Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e)\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec36\" class=\"Section3\"\u003e\n \u003ch2\u003e7.3.10 Stability Studies of NPNLC\u003c/h2\u003e\n \u003cp\u003eThe stability of the optimized NPNLC formulation was evaluated over 6 months under three different storage conditions like 4\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003e0\u003c/sup\u003eC, 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003e0\u003c/sup\u003eC / 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH, and 40\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003e0\u003c/sup\u003eC /75\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH. Drug content and entrapment efficiency were noted every time we increased temperature and time interval, implying also that NPNLC composition remains constant for more than 6 months, as shown in Table \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e. These findings confirm the robust stability profile of the formulation under varied environmental conditions, ensuring its suitability for therapeutic application.(\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e)\u003c/p\u003e\n \u003ctable id=\"Tab6\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eStability studies of NPNLC\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSN\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eOptimized Formulation NLCF2\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTemperature (oC)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInitial\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e4\u0026thinsp;\u0026plusmn;\u0026thinsp;2 oC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e25\u0026thinsp;\u0026plusmn;\u0026thinsp;2 oC/60\u0026thinsp;\u0026plusmn;\u0026thinsp;5%\u003c/p\u003e\n \u003cp\u003eRH\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e40\u0026thinsp;\u0026plusmn;\u0026thinsp;2oC/75\u0026thinsp;\u0026plusmn;\u0026thinsp;5%\u003c/p\u003e\n \u003cp\u003eRH\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e% EE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e73.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72.05\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eDrug Content\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec37\" class=\"Section3\"\u003e\n \u003ch2\u003e7.3.11 Pharmacodynamic study:\u003c/h2\u003e\n \u003cp\u003eIntranasal administered NPNLC reaches the brain cell after crossing BBB through the endocytic process.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e\n \u003ch2\u003e7.3.11.1. Acetic Acid Induced writhing test.\u003c/h2\u003e\n \u003cp\u003eThe pharmacological efficacy of the NPNLC formulation was evaluated using an acetic acid-induced writhing assay in mice, characterized by abdominal constrictions and hind limb stretching. Figure \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e shows a significant reduction in the amount of writhing in the formulation- treated group compared to the control and pure NP groups. A placebo NLC group was used to assess the potential influence of excipients on the pharmacological activity, and no significant effect was observed compared to the control group. Literature suggests that the Naratriptan Drug has to navigate across BBB to reach the somatic afferent terminals of the spinal cord, where it produces a hyperalgesic effect. These findings indicate that the NPNLC formulation effectively permeated the BBB, modulating the trigeminovascular system and reducing the abdominal writhing\u0026rsquo;s and it is used for enhanced therapeutic efficacy.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec39\" class=\"Section3\"\u003e\n \u003ch2\u003e7.3.11.2. Light /dark box model\u003c/h2\u003e\n \u003cp\u003eThe light/dark box model was employed to assess photophobia, which is one of the most common symptoms associated with migraine characterized by an allodynic response to light. Figure\u0026nbsp;13 demonstrates that the optimized NPNLC formulation significantly increased the time spent by Swiss albino mice in the lit compartment compared to the control, placebo and pure NP groups. The placebo NLC group showed no significant effect compared to the control group, indicating that the excipients did not influence the observed pharmacological activity. The result concluded that the NPNLC formulation successfully crossed the blood-brain barrier (BBB) and modulated the activity of the trigeminal nucleus caudalis, ultimately enhancing light tolerance and reducing photophobic responses in treated animals. (\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e)\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec40\" class=\"Section3\"\u003e\n \u003ch2\u003e7.3.11.3. Determination of Brain Uptake Potential\u003c/h2\u003e\n \u003cp\u003eThe developed and validated HPLC technique effectively quantified the drug concentrations in both brain and plasma samples. Result demonstrated a 1.71-fold rise as in drug which obtained a brain from NPNLC once compared with the pure drug. Furthermore, AUC brain / AUC plasma proportion was determined to also be 2.13 and 1.25 after post two hours of administering drugs in the NPNLC and NP, respectively. (Fig. \u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003e,\u003cspan class=\"InternalRef\"\u003e15\u003c/span\u003e,\u003cspan class=\"InternalRef\"\u003e16\u003c/span\u003e,\u003cspan class=\"InternalRef\"\u003e17\u003c/span\u003e). these findings underscore the enhanced brain-targeting capability of NPNLC formulation.(\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e73\u003c/span\u003e)\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"8. Conclusion","content":"\u003cp\u003eThe present study successfully optimized Naratriptan nanostructured lipid carriers (NPNLC) using a Box-Behnken design, focusing on key parameters such as particle size, entrapment efficiency, and % yield. This optimization approach provided valuable insights into the interactions between formulation parameters, which can be explained by particle size, entrapment efficiency, and % yield. The resulting optimized formulation was assessed for the pharmacodynamic study by using acetic acid writhing and light/dark models by comparing with a pure drug which shows remarkable results by comparison. The Quality by Design QbD approach thus proved to be a robust strategy for improving the brain-targeting potential of NPNLC, ultimately leading to enhanced anti-Migraine activity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there was no Funding used for the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Ethics declaration\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal Activity protocols for the optimized formulation were approved by Dr. D. Y. Patil Institute of Pharmaceutical Sciences \u0026amp; Research, Sant Tukaram Nagar, Pimpri, Pune 411018 with Institutional Animal \u0026nbsp; Ethical \u0026nbsp; Approval \u0026nbsp; No \u0026nbsp; DYPIPSR/IAEC/OCT/21-22P-19, in compliance with the guidelines established by the Committee for Control and Supervision of Experiments on Animals (CPCSEA), Government of India. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eHansraj GP, Singh SK, Kumar P. Sumatriptan succinate loaded chitosan solid lipid nanoparticles for enhanced anti-migraine potential. International Journal of Biological Macromolecules. 2015 Nov; 81:467\u0026ndash;76.\u003c/li\u003e\n \u003cli\u003eLade S, Shah N, Burle S. Nanostructured lipid carriers: A vital drug carrier for migraine treatment. Research journal of pharmacy and technology. 2022;15(7):3309-16.\u003c/li\u003e\n \u003cli\u003eHargreaves RJ, Shepheard SL. Pathophysiology of migraine\u0026mdash;new insights. 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Artif Cells Nanomed Biotechnol. 2021;49(1):256-267.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"sumandeep vidypeeth Deemed to be university vadodara Gujrat","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Migraine, Optimization, Naratriptan, solid-lipids, NLCs, Box Behnken Design, Blood-Brain barrier","lastPublishedDoi":"10.21203/rs.3.rs-6029615/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6029615/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMigraine is a prevalent neurological disorder affecting a significant portion of the global population. It is characterized by frequent neurobiological or neurovascular disturbance, including heightened excitability of the central nervous system. Addressing these challenges, this study focuses on developing nanostructured lipids carriers (NLC) for intranasal drug delivery of naratriptan, aiming to provide a rapid and targeted treatment for migraine.\u003c/p\u003e\n\u003cp\u003eThe NLC was successfully formulated, characterized, and optimized using the Box Behnken Design (BBD). The optimized formulations underwent comprehensive characterization including assessments of % yield, encapsulation efficiency (%EE), particle size, Zeta potential, polydispersity index (PDI), surface morphology via scanning electron microscopy (SEM), crystallinity by X-ray Diffraction (XRD), and thermal behavior using differential scanning calorimetry (DSC), stability studies were conducted under varied conditions to evaluate formulation robustness. The optimized NLC formulation (NLCF2) demonstrated promising attributes with a % yield of \u003cstrong\u003e75.65±0.15\u003c/strong\u003e, % EE of \u003cstrong\u003e68.16±1.06\u003c/strong\u003e, particle size of \u003cstrong\u003e79.74±1.37,\u003c/strong\u003e along with PDI of \u003cstrong\u003e0.315\u003c/strong\u003e, and Zeta Potential of \u003cstrong\u003e-32.50.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn-vivo \u003c/em\u003epharmacodynamics studies using acetic acid-induced writhing and light/dark box models revealed significant efficacy of intranasal NLCs. Pharmacokinetic analysis, conducted using high-performance liquid chromatography (HPLC), demonstrated a 1.71-fold increase in drug concentration in the brain for the NLC formulation compared to the pure drug, underscoring enhanced bioavailability and brain targeting.\u003c/p\u003e\n\u003cp\u003eThe successful design and development of intranasal NLCs for migraine treatment represent a novel and effective approach to drug delivery. By facilitating rapid and targeted delivery, these nanocarriers have the potential to minimize systemic side effects and significantly improve the quality of life for individuals suffering from migraines.\u003c/p\u003e","manuscriptTitle":"Advanced Lipid-Based Nanocarriers for Naratriptan Hydrochloride: A Path to Improved Migraine Treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-17 08:17:48","doi":"10.21203/rs.3.rs-6029615/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"345291f0-7f6c-448c-82d0-94ee9cdb3f59","owner":[],"postedDate":"February 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":44336298,"name":"Drug Delivery"},{"id":44336299,"name":"Drug Discovery, Design, \u0026 Development"},{"id":44336300,"name":"Nanoscience"}],"tags":[],"updatedAt":"2025-02-17T08:17:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-17 08:17:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6029615","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6029615","identity":"rs-6029615","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}