Rational Development of Fingolimod Nano-embedded Microparticles as Nose-to-Brain Neuroprotective Therapy for Ischemic Stroke

Rational Development of Fingolimod Nano-embedded Microparticles as Nose-to-Brain Neuroprotective Therapy for Ischemic Stroke | 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 Rational Development of Fingolimod Nano-embedded Microparticles as Nose-to-Brain Neuroprotective Therapy for Ischemic Stroke Xinyue Zhang, Guangpu Su, Zitong Shao, Ho Wan Chan, Si Li, Stephanie Chow, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4715108/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Nov, 2024 Read the published version in Drug Delivery and Translational Research → Version 1 posted 5 You are reading this latest preprint version Abstract Ischemic stroke is one of the major diseases causing varying degrees of dysfunction and disability worldwide. The current management of ischemic stroke poses significant challenges due to short therapeutic windows and limited efficacy, leading to a pressing need for novel neuroprotective treatment strategies. Previous studies have shown that fingolimod (FIN) is a promising neuroprotective drug. Here, we report the rational development of FIN nano-embedded nasal powders using full factorial design experiments, aiming to provide rapid neuroprotection after ischemic stroke. Flash nanoprecipitation was employed to produce FIN nanosuspensions with the aid of polyvinylpyrrolidone and cholesterol as stabilizers. The optimized nanosuspension was subsequently spray-dried into a dry powder, which exhibited excellent redispersibility (RdI = 1.09 ± 0.04) and satisfactory drug deposition in the olfactory region using a customized 3D-printed nasal cast and an Alberta Idealized Nasal Inlet model. The safety of the optimized FIN dry powder was confirmed in cytotoxicity studies with nasal and brain cells, while the neuroprotective effects were demonstrated by observed behavioral improvements and reduced cerebral infarct size in an established mouse stroke model. The neuroprotective effect was further evidenced by increased expression of anti-apoptotic protein BCL-2 and decreased expression of pro-apoptotic proteins CC3 and BAX in brain peri-infarct tissues. Our findings highlight the potential of nasal delivery of FIN nano-embedded dry powder as a rapid neuroprotective treatment strategy for acute ischemic stroke. Nose-to-brain drug delivery Neuroprotection Ischemic stroke Fingolimod Nanoparticles Particle engineering Nasal powder Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction Stroke is a leading cause of permanent disability worldwide, affecting approximately 15 million global citizens annually [ 1 ]. Despite ongoing improvements in stroke care and rehabilitation, many patients still suffer from different degrees of lifelong disabilities. Stroke accounts for an estimated 3–4% of total medical expenses in Western countries [ 1 ], not only affecting patients’ health and quality of life but also imposing a considerable long-term financial burden on healthcare systems. Ischemic stroke is the most prevalent form of stroke and is responsible for around 85% of stroke cases [ 2 ]. Although intravenous thrombolysis and mechanical thrombectomy are clinically available for acute ischemic stroke treatment, only a small portion of patients benefit due to a restricted therapeutic time window, resulting in a high rate of disability among stroke patients [ 2 ]. Consequently, there is a need to develop rapid and effective neuroprotective therapies for the management of acute ischemic stroke. Upon the onset of ischemic stroke, blood supply to the neurons is interrupted immediately, resulting in substantial cell death [ 3 ]. Timely treatment has a significant impact on relieving the severity of the patient's disease condition and their degrees of lifelong disabilities. However, it typically takes several hours for oral neuroprotective medications to reach their optimal treatment concentration in the brain. Hence, alternative drug administration methods with faster onset are required, especially for patients who are unable to take medicines orally during an acute ischemic stroke episode. Nose-to-brain drug administration has become attractive nowadays because of its minimal invasiveness, easier self-administration, higher effectiveness with less systemic side effects, and more direct route to the central nervous system [ 4 , 5 ]. Previous research has indicated the drug can be delivered to the brain rapidly after intranasal administration [ 6 ]. Integrating intranasal administration with nanotechnology further enhances the potential of this delivery approach, as it prolongs drug residence time at the absorption site, increases cellular internalization, and regulates the release of encapsulated drugs [ 7 ]. Previous studies have also demonstrated that intranasal delivery of drug nanoparticles could achieve improved bioavailability [ 8 ] and high brain targetability [ 9 ] while concurrently reducing off-target concentration in the bloodstream [ 10 ]. Moreover, drug encapsulation within nanoparticles can minimize degradation risks [ 11 ] and promote rapid brain uptake [ 12 , 13 ]. The above-mentioned properties are highly sought after for the intranasal administration of neuroprotective nanoparticles, especially for targeted transport to the ischemic brain. Compared with nasal sprays, nasal powders possess unique benefits such as extended retention time and superior stability against enzymatic degradation within the nasal cavity [ 14 , 15 ]. Furthermore, nasal powders can be reconstituted in an aqueous buffer as a nasal solution or suspension to offer more flexible dosing according to specific clinical needs [ 16 ]. A notable obstacle in the engineering of nanoparticle-loaded powders is the maintenance of appropriate redispersibility of dry powder back into the nanoparticle once contact with the nasal fluid to preserve the therapeutic merits of nanoparticles. Another major challenge in fabricating dry powders for optimal nose-to-brain delivery lies in particle size control, as only particles with a diameter of around 10 µm can achieve greater deposition in the olfactory region, i.e., the primary target believed to be responsible for nose-to-brain delivery [ 17 , 18 ]. In the present study, spray drying was selected for converting nanosuspensions into dry powders owing to its commercial availability for simplified scale-up, as well as its ability to tailor particles for targeted intranasal delivery to the olfactory region [ 19 , 20 ]. Fingolimod (FIN) is an oral drug approved for the treatment of multiple sclerosis [ 21 ]. It has also demonstrated neuroprotective effects in various animal studies and clinical trials. For instance, ischemic stroke models of mice that received FIN intravenously or intraperitoneally showed a decrease in infarct size and improved behavioral testing results [ 22 , 23 ]. Moreover, stroke patients treated with standard management and oral FIN medication beyond the 4.5 h treatment window for intravenous thrombolysis (tissue plasminogen activator) displayed reduced secondary tissue injury, decreased neurological impairments, and improved post-stroke recovery compared to patients who received standard management alone [ 24 ]. Consequently, FIN is a promising neuroprotective drug with therapeutic potential for acute ischemic stroke. Curcumin (CUR), a polyphenol found in turmeric, has also shown neuroprotective effects in both hemorrhagic and ischemic stroke cases [ 25 ]. However, the delivery of FIN and CUR to the brain still requires optimization for improved clinical outcomes. While separate studies have shown the therapeutic potential of FIN for acute ischemic stroke, no research has yet studied its neuroprotective effect through intranasal nanotherapy. This delivery strategy confers the unique advantage of rapid treatment for patients during ambulance transportation, obviating the requirement for conscious patient cooperation. Hence, the objective of this study was to develop FIN nano-embedded nasal powders for rapid neuroprotection after the onset of acute ischemic stroke. To this end, a full factorial design of experiments was conducted to examine the effects of and optimize critical formulation and processing parameters for FIN nanosuspension and its nano-embedded dry powder formulations. The optimized powder formulation was characterized for various pharmaceutical properties, such as aqueous redispersibility, particle size, nasal deposition profile, crystallinity, cytotoxicity, and stability. Lastly, the neuroprotective effects of nasally administered FIN nanoparticles were evaluated through neurological functional tests and infarct size measurements in a well-established acute ischemic stroke model. 2. Materials and Methods 2.1 Materials Fingolimod (FIN, > 99.9% purity) was purchased from Hefei Hirisun Phamatech (Hefei, China). Curcumin (CUR, > 99.5% purity) was sourced from Yung-Zip Chemicals (Taichung, Taiwan). Methanol (MeOH) and ethanol (EtOH) were obtained from VWR BDH Chemicals (VWR International S.A.S., Fontenay-sous-Bois, France). Ultra-purified water (UPW) was generated using a Barnstead NANOpure Diamond system (Thermo Fisher Scientific, Waltham, MA, USA). Polyvinylpyrrolidone (PVP K30) and cholesterol (CLT) were procured from Sigma-Aldrich (St. Louis, MO, USA), and Mannitol (Pearlitol 160C) was purchased from Roquette (Lestrem, France). Trifluoroacetic acid (TFA), Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12), Dulbecco’s Modified Eagle’s Medium (DMEM), Minimum Essential Medium (MEM), Kaighn's Modification of Ham's F-12 Medium (F-12K medium), 0.25% (w/v) trypsin-EDTA, phosphate-buffered saline (PBS, 10×), fetal bovine serum (FBS), and antibiotic–antimycotic (100×) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Additionally, a variety of cell lines were obtained from the American Type Cultural Collection (ATCC; Manassas, VA, USA), including PC 12 cells, RPMI 2650 cells, Calu-3 cells, and SH-SY5Y cells. Nissl Staining Solution (Cat. No. C0117) was purchased from Shanghai Beyotime Biotechnology Co., Ltd (Shanghai, China). Polyvinylidene difluoride membrane was purchased from Bio-Rad (California, USA). Primary antibodies against Cleaved Caspase-3 (CC3) (Cat. No. 9664) and β-Tubulin proteins (Cat. No. #2146S) were purchased from Cell Signaling Technology (Massachusetts, USA) whose t against B-cell lymphoma 2 (BCL-2) (Cat. No. WL01556) and BCL-2 associated X (BAX) (Cat. No. WL01637) proteins were purchased from Wanlei Bio (Liaoning, China). Horseradish peroxidase-labeled goat anti-rabbit IgG(H + L) secondary antibodies (Cat. No. A0208) were purchased from Beyotime (Shanghai, China). 2.2 High-Performance Liquid Chromatography (HPLC) FIN and CUR were assayed by HPLC using a C18 column (5 µm, 250 mm × 4.6 mm; Eclipse Plus; Agilent Technologies, Lexington, MA, US) coupled with a guard column (5 µm, 12.5 mm × 4.6 mm) and a photodiode array detector (Infinity 1260 LC System, Agilent Technologies, Lexington, MA, US) under a gradient mode ( Table S1 ). The mobile phase comprised mobile phase A [0.15% TFA in UPW (v/v)] and mobile phase B [0.15% TFA in MeOH (v/v)]. A 20 µL sample was injected into the column at a flow rate of 1 mL/min. The UV detection wavelengths of FIN and CUR were set at 220 nm and 430 nm, respectively. FIN and CUR eluted at around 9.6 min and 5.3 min, respectively. The calibration curves showed excellent linearity for both FIN ( R 2 = 0.9997) and CUR ( R 2 = 0.9999). 2.3 Preparation of FIN Nanosuspensions The FIN nanosuspension was produced using a four-inlet multi-inlet vortex mixer (MIVM) [ 26 ], as illustrated in Fig. 1 and Video S1 . Specifically, the drugs FIN and CUR, along with CLT, were dissolved in EtOH as an organic stream in inlet 1, while the remaining inlets (inlets 2–4) were loaded with PVP aqueous solution. The flow rates of inlets 2 and 4 were regulated by a PHD ULTRA syringe pump (Harvard Apparatus, Holliston, MA, USA) at 99 ml/min, and inlets 1 and 3 were controlled by another syringe pump (Terumo Corporation, Tokyo, Japan) at 11 ml/min. The FIN nanosuspension was collected from the outlet stream of the MIVM. The flow pattern can be characterized by calculating the Reynold number (Re), as shown in Eq. 1. The resulting Re was fixed around 4,000 to ensure homogeneous mixing of the four inlet streams prior to nanoprecipitation [ 27 , 28 ]. $$\:Re=\sum\:_{i=1}^{4}\frac{{\rho\:}_{i}{v}_{i}d}{{\mu\:}_{i}}\:=\:\frac{4}{\pi\:D}\sum\:_{i=1}^{4}\frac{{\rho\:}_{i}{Q}_{i}}{{\mu\:}_{i}}\:\:\:\:\:\:\:\:\:\:\:\left(Equation\:1\right)$$ where i is the stream number, ρ i is the fluid density (kg/m 3 ), Q i is the stream flow rate (m 3 /s), µ i is the fluid viscosity (Pa´s), and D is the internal diameter (m) of the MIVM. 2.4 Characterization of FIN Nanosuspensions 2.4.1 Particle Size, Size Distribution and Zeta Potential The z -average particle size, size distribution, and polydispersity index (PDI) of FIN nanoparticles were measured by dynamic light scattering (DLS) using a Delsa Nano C particle analyzer (Beckman Coulter, Brea, CA, USA). The viscosity and refractive index of the medium were assumed to be the same as those of pure water (0.89 mPa·s and 1.331 at 25°C). The zeta potential of FIN nanoparticles was determined using the same particle analyzer mentioned above. The measured electrophoretic mobility was converted into zeta-potential using the Smoluchowski relationship [ 29 ]. 2.4.2 Physical Stability The physical stability of FIN nanosuspension was monitored by measuring the change in particle size over time [ 30 ]. The test was terminated when either a > 20% change in particle size occurred or visible precipitation was observed in the nanosuspension. 2.4.3 Encapsulation Efficiency (EE) and Drug Loading (DL) of the Nanosuspension The determination of EE and DL was performed using an established protocol [ 31 ]. Briefly, 15 mL of FIN nanosuspension was transferred into an Amicon® Ultra 30kDa centrifugal filter device (Sigma Aldrich, St Louis, MO, USA) and centrifuged at 4000 x g for 40 minutes. The filtrate, containing free FIN and CUR, was collected for HPLC analysis while the concentrated sample was freeze-dried using a Freezone 6 Liter Benchtop Freeze Dry System with Stoppering Tray Dryer (Labconco Corporation, Kansas City, MO, US). The freeze-dried product was weighed and dissolved in a mixture of UPW and MeOH with 0.15% TFA [23:77 ( v/v )] for HPLC analysis of the drug content in the nanoparticles. The EE and DL were then calculated according to Equations 2 and 3, respectively. $$\:\text{E}\text{E}\:\left(\%\right)=\frac{total\:amount\:of\:drug-amount\:of\:free\:drug}{total\:amount\:of\:drug}\times\:100\%\:\:\:\:\:\:\:\:Equation\:\left(2\right)$$ $$\:D\text{L}\:\left(\%\right)=\frac{total\:amount\:of\:drug\:in\:nanoparticles}{total\:amount\:of\:nanoparticles}\times\:100\%\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:Equation\:\left(3\right)$$ 2.4.4 Transmission Electron Microscopy (TEM) A single drop of freshly produced FIN nanosuspension was dripped on a carbon film TEM grid that had been discharged using the PELCO easiGlow™ Glow Discharge Cleaning System (Redding, CA, US), followed by staining with 2% ( v/v ) uranyl acetate for 1 minute. Then, the Tecnai™ G2 20 S-TWIN Transmission Electron Microscope (FEI, Hillsboro, OR, US) was employed to image the nanoparticles on the air-dried grid at ×19,500 magnification. 2.5 Design of Experiment (DoE)-guided Optimization of FIN Nanosuspension A 2-level, 3-factor full factorial design was employed to examine the main effects and interactions of selected formulation parameters on the critical quality attributes (CQAs) of the FIN nanosuspension ( Table S2 ). The processing parameters were the initial concentration of FIN (mg/ml) (A), the mass ratio between CLT and FIN (w/w) (B), and the concentration of PVP solution [% (w/v)] (C), while the responses included particle size (nm) (Y 1 ), PDI (Y 2 ), physical stability (hours) (Y 3 ), and EE of FIN (%) (Y 4 ). The initial concentration of CUR was fixed at 2.5 mg/mL. The levels of each variable were set as + 1, 0, and − 1, and a total of 9 experimental runs (2 3 + 1 runs as the center point) were performed. The optimal processing parameters of FIN nanosuspension were identified using the desirability function approach based on the regression models constructed by Design-Expert 13 for the responses [ 32 ]. Each response was associated with a partial desirability function ( d ), where a fully desired response was assigned a value of 1 and an unfavorable response was assigned a value of 0. The overall desirability value ( D ) was determined using the geometric mean of the partial desirability functions. 2.6 Preparation of FIN Nano-embedded Microparticle Dry Powder Formulations FIN nano-embedded microparticle dry powder formulations were produced using spray drying. Specifically, mannitol solution was mixed with freshly prepared optimized FIN nanosuspension, and this mixture was fed into a Büchi spray dryer (Mini Spray Dryer B-290 coupled with Dehumidifier B-296, Flawil, Switzerland) with nitrogen as the drying gas [ 31 ]. The aspiration rate and inlet temperature were fixed at 100% (38 m 3 /h) and 110 ℃, while the atomization flow rate and feed rate were varied in each run according to the experimental design. The resulting product was collected in a Falcon-50 ml conical tube and transferred to a desiccator upon spray drying. 2.7 Characterization of FIN Nano-embedded Microparticle Dry Powder Formulation 2.7.1 Aqueous Redispersibility The redispersibility test was performed by reconstituting the spray-dried powder in UPW at room temperature. Briefly, 15 mg of dry powder was transferred into 10 ml of UPW, and the resulting suspension was stirred at 75 rpm for 10 minutes. After 3 minutes, the particle size was measured as described in Section 2.4.1 . The redispersibility index (RdI) was denoted as S f /S i , where S i and S f represent the particle size of FIN nanoparticles before and after spray drying, respectively [ 33 ]. A RdI value of 1 indicates that the particle size remains unchanged during the spray drying process. 2.7.2 Particle Size Distribution by Laser Diffraction A HELOS/KR laser diffractometer (Sympatec, Germany) was used to determine the volumetric size distribution of the dry powder, as previously reported with minor modifications [ 34 ]. Briefly, a Unidose powder nasal spray system (Aptar Pharma, France) filled with 5.0 ± 0.5 mg dry powder was connected to the laser diffractometer (30 ◦ angle) using an adaptor. The dry powder was then dispersed at a flow rate of 15 L/min. The spherical volume diameters at 10% (D 10 ), 50% (D 50 ), and 90% (D 90 ) cumulative volumes were recorded. The span of the dry powder was expressed as (D 90 − D 10 )/ D 50 . Each sample was measured three times. 2.7.3 In Vitro Evaluation of Nasal Aerosol Depositions 2.7.3.1 3D-printed Nasal Cast Model As depicted in Fig. 2 a, a Next Generation Impactor (NGI) (Copley, Nottingham, UK) coupled with a customized 3D-printed nasal cast model was employed to assess the in vitro aerosol performance of the optimized FIN dry powder formulation [ 35 ]. The 3D-printed nasal cast can be dismantled into two parts: the olfactory region and the respiratory region. A thin layer of silicon grease (Slipicone; LPS Laboratories, Tucker, GA, USA) was sprayed on all stages of the NGI to minimize particle bouncing. An unidose powder nasal spray device (Aptar Pharma, France) was used to disperse 15.0 ± 1.0 mg of optimized FIN dry powder into the nasal cavity under inspiratory flow rates of 0, 7.5, and 15 L/min. The insertion depth and the insertion angle of the nasal device into the nostril were 5 mm and 60 ◦ from the horizontal plane, respectively. After dispersion, the powder in the 3D-printed nasal cast model and NGI stages was rinsed using the HPLC mobile phase (UPW and MeOH with 0.15% TFA [23:77 (v/v)]). The resulting solution was filtered through a 0.45-µm membrane for FIN assay. The deposition experiment was repeated thrice. The recovered dose was defined as the total mass of FIN recovered in the 3D-printed nasal cast model and the NGI stages, and all fractions were calculated with respect to the recovered dose. The nasal device fraction was calculated with the powder mass remaining in the nasal spray device after dispersion, while the throat fraction was calculated using the powder mass deposited in the throat and adaptor regions connecting the NGI and nasal cast. The NGI stages fraction was calculated based on the powder mass deposited in Stages 1–7 and the MOC of the NGI. 2.7.3.2 Alberta Idealised Nasal Inlet (AINI) Model An Alberta Idealised Nasal Inlet (AINI) (Copley, Nottingham, UK) coupled to the NGI was also utilized to examine in vitro nasal aerosol deposition (Fig. 2 b) [ 4 ]. This model was created based on a set of realistic nasal anatomies and composed of four detachable components, i.e., olfactory region, vestibule, turbinates, and nasopharynx [ 36 – 39 ]. The procedures for coating the NGI stages, filling the nasal spray device, inserting the nasal spray device, FIN assay, and calculations of the recovered dose and nasal device fraction were the same as those described in Section 2.7.3.1 . 2.7.4 Scanning Electron Microscopy A Hitachi S-4800 FEG field emission scanning electron microscope (Hitachi, Tokyo, Japan) was utilized to characterize the particle morphology of the spray-dried powder at 5.0 kV. The powder sample was gently dispersed on carbon tape mounted on a SEM stub and subsequently coated by an ~ 11 nm gold-palladium alloy using a sputter coater for 90 seconds to prevent charging interferences during the imaging. 2.7.5 Powder X-Ray Diffractometry (PXRD) A Rigaku SmartLab 9 kW diffractometer with a copper rotating anode (K alpha1 1.54059 Å, K alpha2 1.54441 Å) rated at 160 mA/ 45 kV was used to collect the PXRD patterns of the samples. A K beta nickel filter was used to filter the diffraction signals. Each sample was scanned within a 2θ range of 3° to 40°, with a step width of 0.02° and a scanning speed of 5.0° per minute. 2.7.6 Fourier-Transform Infrared Spectroscopy (FTIR) A Spectrum Two FTIR spectrometer (Perkin Elmer, Waltham, MA, USA) was used to generate FT-IR spectra in KBr diffuse reflectance mode. The scan was performed in the range of 4,000 cm − 1 to 1,000 cm − 1 at intervals of 0.5 cm − 1 . A total of 32 scans were performed at a resolution of 4 cm − 1 for each sample. 2.7.7 Differential Scanning Calorimetry (DSC) The thermal characteristics of the samples were measured using a DSC 250 differential scanning calorimeter (TA Instruments, New Castle, DE, USA). Prior to the measurement, pure indium was used for calibration. Each sample (3 ± 0.5 mg) was encased in a Tzero hermetic pan and heated from 50°C to 250°C at a ramp rate of 10°C/min under an N 2 flow rate of 20 mL/min. 2.7.8 Thermogravimetric Analysis (TGA) The residual solvent and moisture content ( M ) of the spray-dried powder was determined using a TGA550 thermogravimetric analyzer (TA Instruments, Newcastle, DE, USA) according to Eq. (5). Approximately 3 mg of sample was loaded in a platinum pan and heated from 25°C to 200°C at a scanning rate of 10°C/min under a N 2 flow rate of 20 mL/min. $$\:M\:\left(\%\right)=\frac{{m}_{0}-{m}_{1}}{{m}_{0}}\times\:100\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:Equation\:\left(4\right)$$ where m 0 and m 1 represent the weight of the measured sample before and after the experiment, respectively. 2.7.9 Encapsulation Efficiency (EE) and Drug Content of Powders To determine the encapsulation efficiency of reconstituted nanosuspension, 20 mg of spray-dried dry powder was dispersed into 10 ml of UPW under a stirring rate of 75 rpm for 10 minutes. The reconstituted nanosuspension was subsequently transferred into the filter device (Amicon® Ultra-15, Sigma Aldrich, St Louis, MO, USA) and centrifuged at 4000 x g for 40 min. The analytical procedure was the same as mentioned in section 2.4.3 . Regarding the drug content of FIN and CUR in the spray-dried dry powder, 10 mg of sample was accurately weighed and transferred into the tube and dissolved in a 2 ml mixture of UPW and MEOH with 0.15% TFA [23:77 ( v/v )] for HPLC assay of FIN and CUR in the sample. 2.7.10 Stability Studies The samples were stored in screw-capped glass tubes at 4°C, room temperature, and 40°C under 30% relative humidity for 2 months. The chemical stability of the sample was monitored by conducting HPLC assays of FIN and CUR, while the physical stability of the sample was checked by the DSC, TGA, and PXRD analysis. 2.7.11 In Vitro Drug Release The drug release profiles of the samples were obtained using a reported protocol with modification [ 40 ]. 50 ± 0.5 mg of powder samples or its physically mixed counterpart was dispersed into 20 ml of simulated nasal fluid, and the resulting solution was stirred at 75 rpm for 3 hours at 34 ± 0.1°C [ 41 ]. 0.5 ml of solution was withdrawn at designated time points (15, 30, 60, 120, and 180 minutes) and transferred into an Amicon® Ultra-0.5 centrifugal filter unit. Medium replacement was carried out upon each sample collection. The FIN content in the filtrate was assayed by HPLC. The experimental procedure was repeated with the change of the medium to a mixture of simulated nasal fluid and ethanol [80:20 (v/v)]. 2.8 DoE-guided Optimization of FIN Nano-embedded Dry Powder Formulation The experimental design, optimization, and model validation of the fabrication of FIN dry powder formulation were the same as the FIN nanosuspension production described in Section 2.5 . Herein the formulation and processing parameters in the DoE ( Table S.3. ) included the ratio between mannitol and nanoparticle (w/w) (A), the atomization gas flow rate (L/h) (B), and the feed rate of solution during the spray drying process (ml/min) (C), while the responses included redispersibility of the dry powder (Z 1 ), PDI of the reconstituted nanosuspension (Z 2 ), and volumetric particle size of dry powder (µm) (Z 3 ). Again, the optimal processing parameters of FIN co-loaded dry powder were identified by the desirability function approach based on the regression models constructed by Design-Expert 13 for the responses. 2.9 In Vitro Cellular Study 2.9.1 Cell Culture SH-SY5Y cells were cultured in DMEM culture medium, and PC 12 cells were cultured in F-12K nutrient mixture medium containing L-glutamine. RPMI 2650 cells were cultured in MEM, while Calu-3 cells were cultured in DMEM/F12 culture medium. These cell culture mediums were supplemented with 10% (v/v) FBS and 1% (v/v) antibiotic-antimycotic. The cells were cultured in a humidified incubator at 37°C and 5% CO 2 and were passaged in different ratios when the cell density reached 80% confluence. 0.25% trypsin-EDTA was used to detach the cells from the T75 flasks. 2.9.2 In Vitro Cellular Viability Assay Cells were seeded in 96-well plates at a density of 2 × 10 4 cells/well (for RPMI 2650 and PC-12), 0.5 × 10 5 cells/well (for SH-SY5Y), or 1 × 10 5 cells/well (for Calu-3) and cultured in a humidified incubator at 37°C and 5% CO 2 overnight. On day 2, the cells were treated with reconstituted optimized FIN dry powder, raw FIN, and a physical mixture of FIN powder formulation components at FIN concentrations ranging from 2.5 to 1000 nM for 24 hours. The reconstituted optimized FIN dry powder was prepared by redispersing optimized FIN dry powder in the cell culture medium. Raw FIN and their physical mixture were dissolved in DMSO and further diluted by the cell culture medium to a desired concentration. Considering the cytotoxicity of DMSO, cells were also treated with raw DMSO, which was the same as the DMSO concentration in the treatment groups as the control group. After 24 hours, the cells were incubated with 100 µL of cell culture medium with 10% CCK-8 solution for 3 hours. The spectrophotometric absorbance of the sample was measured at 450 nm using a microplate spectrophotometer (MultiSkan Go, Thermo Fisher Scientific, Waltham, MA, USA). The cell viability was calculated as the percentage of the absorbance from the treatment groups divided by the absorbance from the control group. All measurements were performed in triplicate. 2.10 In Vivo Animal Study 2.10.1 Animals and Ethics Statements All animal studies followed the ethical policies and guidelines recommended by ARRIVE (Animal Research: Reporting of In Vivo Experiments) and National Research Council's Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of the Animal Experimentation of Jinan University (Approval No.: 20230901-0012). Male C57BL/6 mice (6–8 weeks old) were obtained from Zhuhai Baishitong Biotechnology Co., Ltd and were used for the ischemic stroke models. All mice were kept in a daily cycle of 12 hours of light and 12 hours of darkness with humidity and temperature controls. Water and food were freely available to the mice at all times in their cages. 2.10.2 Development of the Ischemic Stroke Model Electrocoagulation was used to create the middle cerebral artery occlusion (MCAO) model based on a reported protocol on day 1 [ 42 ]. Mice were anaesthetized using 1% (w/v) pentobarbital sodium at a dose of 50 mg/kg by intraperitoneal injection. Subsequently, a skin incision was made between the ear and right eye to expose the skull and temporalis muscle. The middle cerebral artery was exposed by penetrating the skull with a microdrill. Finally, a small vessel electrocoagulator was utilized to cauterize the distal middle cerebral artery. Following the restoration of the temporalis, a surgical suture was used to close the wound in the head skin. 2.10.3 Animal Grouping A total of 32 mice were randomly divided into 4 groups (n = 8 per group): (1) sham-operated group (Sham group); (2) MCAO group receiving 0.9% saline solution intranasally (IN) (MCAO group); (3) MCAO group treated with the optimized FIN dry powder (1 mg/kg of FIN) reconstituted in 0.9% saline solution via intravenous (IV) injection (MCAO + IV FIN group); and (4) MCAO group treated with the optimized FIN dry powder (1 mg/kg of FIN) reconstituted in 0.9% saline solution (~ 15 µL) intranasally (IN) (MCAO + IN FIN group). The initial dose was administered 30 minutes following the ischemic stroke surgery, and the second dose was given 24 hours post-surgery. On the third day, the mice were sacrificed, and brain tissues were collected for western blot analysis. 2.10.4 Behavioural Assessments and Neurological Function Evaluation The neurological recovery of mice was assessed using the adhesive removal test balance beam test, and rotarod test on days 2 and day 3 [ 43 – 45 ]. Baseline behavioural assessment was conducted one day prior to the stroke surgery. In the adhesive removal test, mice were placed in a container, and the time for the mice to remove the adhesive tapes from the bilateral paws was recorded. For the balance beam test, the time taken for the mice to walk across a beam (1.20 m in length, 1.0 cm in width, and 0.7 m in height) was recorded after training. In the rotarod test, mice were trained at constant speed three times before the surgery and the retention time for mice to remain on the rotarod with accelerated speed (starting at 4 rpm and increasing by 46 rpm per 60 seconds until the 50 rpm) was assessed. Furthermore, the Longa score was used to assess the neurological deficits of mice on day 3, especially the motor function of mouse [ 46 , 47 ]. The weight of mice was monitored daily. 2.10.5 Histological Assessment The histological assessments were conducted on day 2 after the behavioural assessments and neurological function evaluation as previously described [ 48 ]. Briefly, the mice were anesthetized (1% pentobarbital sodium, 50 mg/kg) and were perfused with 0.9% sodium chloride transcardially. After fixing with 4% paraformaldehyde, the brain was removed rapidly and dipped into the wax, followed by embedding and sectioning in 4 µm thickness. The brain slice was observed at the posterior and anterior levels based on Paxinos and Franklin's mouse brain in stereotaxial coordinates. The brain slices were defatted by xylene and dehydrated by alcohol. For Nissl staining, the brain slices were immersed in 0.1% cresol violet for 30 minutes at room temperature. The brain slices were dehydrated after rinsing by UPW, followed by transparentizing and sealing with neutral gum. The TissueFAXS PLUS system from TissueGnostics was employed to scan slides automatically. 2.10.6 Magnetic Resonance Imaging (MRI) The MRI was conducted on day 2 using a reported protocol with modification [ 49 ]. The 9.4 T small animal MRI scanner (Bruker PharmaScan) was used to obtain MRI images of the mice 24 hours after the stroke surgery. The parameters of the T2-weighted imaging (T2WI) imaging were set as a field of view (FOV) = 20 × 20 mm, 17 axial slices with a slice thickness of 1 mm, a matrix of 256 × 256 and 2D fast-spin echo sequence (3500/33 ms of repetition time/echo time, 2 average). After anesthetization, the respiratory rate and the temperature of mice were monitored during the imaging process. The machine was placed over the brains of mice, followed by the scanning by T2WI imaging and quantitively analysis using a 3D slicer software. Based on the T2WI images, the 3D slicer was used to reconstruct the 3D images. The infarct and non-infarct zones were separated using threshold modification. 2.10.7 Western Blot Analysis The western blot analysis was conducted on day 3 according to a reported protocol with modifications [ 43 ]. The RIPA buffer was used for lysing and extracting proteins from the mouse brain. Protein samples (5–30 µg/lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene difluoride membrane (Bio-Rad). The membrane was blocked using TBST buffer containing 5% milk for one hour, followed by overnight incubation with primary antibodies against CC3, BCL-2, BAX, and β-Tubulin (as internal control) proteins at 4℃ in 5% milk-TBST. The blot was then washed and incubated with secondary antibodies in TBST buffer containing 5% milk for one hour at room temperature. The ChemiDoc Touch imaging system (Bio-Rad) was used to detect the protein bands, while Image Lab software and Tanon 2500 Gel Imaging System were employed for data analysis. Protein bands were analyzed and quantified using Quantity One (Bio-Rad) or ImageJ with normalization provided by the respective loading controls in the same samples. 2.11 Statistical Analysis Data were shown as mean ± standard deviation (S.D.). The optimal processing parameters of FIN nanosuspension and dry powder were obtained by Design-Expert (Version 21.3.1; Stat-Ease, Inc., Minneapolis, USA) using least-square regression and nested ANOVA of the responses. Trios (Version 5.1.1; TA Instrument, New Castle, DE, USA) was used for the analysis of DSC data. A p -value < 0.05 was considered statistically significant. 3. Results and Discussion 3.1 Optimization of FIN Nanosuspensions The selection of formulation parameters and the corresponding design space ( Table S2 ) in the factorial design for fabricating FIN nanosuspensions were guided by preliminary data and technical considerations for nose-to-brain drug delivery. Without the aid of stabilizers, the resulting nanosuspensions exhibited visible precipitation within 5 minutes right after production, while the incorporation of PVP and CLT into the formulations significantly enhanced the physical stability of nanosuspensions ( Table S4 ). Regarding the response specifications, as the diameter of olfactory axon in humans falls within a typical range of 100–700 nm, and previous research has demonstrated that nanoparticles with a size of less than 200 nm can be transported effectively to the brain via transcellular neuronal absorption, the target size for FIN nanoparticles was set in the range of 0-200 nm [ 50 , 51 ]. In addition, considering that the nanosuspensions would be spray-dried into dry powders, they should remain colloidally stable before the drying process. Therefore, the physical stability was set as over 24 hours, allowing sufficient transit time in real manufacturing practice. To unleash the therapeutic potential of the formulations, targets for the EE of FIN and PDI of the nanoparticles were set in line with the literature standards at 90% and < 0.3, respectively [ 31 , 52 ]. Although single-factor experiments are frequently used for optimizing drug formulations and manufacturing processes, it is relatively time-consuming and incapable of identifying potential interaction effects of factors on responses [ 53 , 54 ]. Consequently, a full factorial DoE was employed in this study. Table 1 presents the physicochemical properties of FIN nanosuspensions obtained from the DoE. The particle size of the nanosuspensions ranged from 107.4 ± 5.4 nm to 187.2 ± 2.4 nm, which were all within the target range. The PDI of these formulations, except for NF2, satisfied the pre-set requirement of < 0.3. Notably, there was a discrepancy in the stability among these formulations. For example, NF2 and NF5 showed precipitation within 30 minutes, whereas NF3 remained stable for at least 72 hours. The EE of FIN among these formulations varied from 60.51 ± 1.84% to 95.73 ± 0.04%, while the EE of CUR consistently surpassed 99.99%. The coded regression equations for each response model of the FIN nanosuspensions are shown in Table S5 . These models were tested by two-way nested ANOVA and displayed statistical significance ( p < 0.0001) for all responses, suggesting that the factors and their interactions were significantly correlated with the responses. They were also considered reliable with predicted R ² values close to or exceeding 0.85, which agreed with the adjusted R ² values. All regression models were therefore used to probe the relative effects of factors and their interactions on the responses. The Pareto charts in Fig. 3 show the standardized effects of factors and their interactions on the responses. All studied factors and their interactions exerted a significant effect on the particle size of FIN nanosuspensions (Y 1 ). Specifically, increasing the initial FIN concentration (A), CLT: FIN ratio (B), the concentration of PVP solution (C), and their interaction terms AC and BC tended to produce larger particles. The observations in the main effects could be partly explained by the nucleation and growth theories. Upon bulk mixing of the aqueous and organic streams in the MIVM, an upsurge in the supersaturation of hydrophobic solutes (FIN, CUR, and CLT) would concurrently increase their nucleation and particle growth rates, resulting in a contrasting effect on the particle size. Quenching further particle growth requires adequate deposition of the water-soluble stabilizer (PVP) on nanoparticles (i.e., nuclei with dimensions beyond the critical size) via molecular diffusion, which is a time-dependent process [ 55 ]. This explains why a sole rise in factor A or B led to an increase in particle size, as the resulting nanoparticles were not instantly stabilized by PVP, and their effects on particle growth dominated that of nucleation. It is worth noting that factor C could influence particle size in both negative and positive ways. While PVP could inhibit nanoparticle growth and aggregation by surface capping, it also acted as a solubilizing agent to alter the supersaturation levels of FIN and CLT in the systems and, consequently, their nucleation kinetics. The latter effect was more apparent in the current design space. The dual roles of PVP also caused factor C to have a lower t -value in two-way nested ANOVA and coefficient in the regression equation compared to the other main factors. Surprisingly, the interaction terms AB and ABC, albeit marginally statistically significant, were negatively correlated with particle size. We speculate that when both factors A and B increased, the precipitated hydrophobic CLT was able to shorten the diffusion path and time of PVP from the bulk solution to the nuclei, thereby yielding smaller nanoparticles. However, the molecular interplays in nanoparticle formation and stabilization for a multi-component system are complex, and further research (e.g. using molecular dynamics simulation) is warranted to delineate their exact mechanisms. Pertaining to the PDI (Y 2 ), physical stability (Y 3 ), and EE of FIN (Y 4 ) responses, the factor-response relationships elucidated from the models generally aligned with the findings derived from the response model of particle size (Y 1 ) and our expectations. As mentioned earlier, augmenting factor A or B would elevate the supersaturation level of the hydrophobic solutes, thereby facilitating the entrapment of more FIN molecules within the nanoparticle core during the FNP process. The resulting larger nanoparticles also possessed lower surface energy, which served to minimize particle aggregation and produce a more monodisperse nanosuspension. It is not surprising that factor C and its related 2-factor interactions (i.e., AC and BC) displayed erratic effects on the PDI, EE, and stability since PVP serves multiple functions as a crystallization inhibitor, solubilizer, and stabilizer in the nanosuspension formulation as aforementioned. Briefly, factor C had a positive, negative, and insignificant effect on PDI, EE, and stability, respectively, but its interaction factors AC and BC showed divergent trends (AC: negative for PDI, positive for EE and stability; BC: insignificant for PDI and stability, positive for EE). It should be noted that these regression models are of good predictive ability within our design space but their applicability to other systems may be limited, particularly if the design spaces of those systems differ significantly from the present study. The regression models were simplified by eliminating non-statistically significant terms (Table 2 ), and the desirability function was applied to optimize the FIN nanosuspension, and the highest desirability value (0.951) was obtained under the condition of A = 5 (mg/mL), B = 1.5 ( w/w ) and C = 0.25 (% w/v ). Additional experimental runs ( n = 3) using such parameters were performed for model validation. The resulting nanosuspensions had a particle size of 134.0 ± 0.6 nm ( Fig. S1 a ), PDI of 0.179 ± 0.021, physical stability of 72 ± 0 hours, and EE of FIN of 90.67 ± 0.08%, which were all in line with the predictions (particle size of 133.83 nm, PDI of 0.161, physical stability of 72 hours, and EE of FIN of 91.07%). The DLs of FIN (8.00 ± 0.25%) and CUR (4.43 ± 0.15%) were well aligned with the theoretical values, i.e., 8% for FIN and 4% for CUR. The zeta potential of the optimized nanosuspension was − 0.24 ± 0.24 mV, suggesting its near-neutral state. As shown in Fig. S1 b , FIN nanoparticles displayed roughly spherical morphology with similar size to that of DLS. As a result, the optimized FIN nanosuspension was used for the subsequent development of nano-embedded microparticle dry powders. Table 1 Physicochemical properties of FIN nanosuspensions prepared based on the 3-factor 2-level factorial design (n = 3). Nanosuspension Formulation (NF) Factors with levels Responses and results A B C Particle Size (nm) (Y 1 ) PDI (Y 2 ) Physical Stability (hours) (Y 3 ) EE of FIN (%) (Y 4 ) NF1 5 0.5 0.25 136.6 ± 1.3 0.238 ± 0.008 0.7 ± 1.2 88.87 ± 1.25 NF2 5 0.5 1 107.4 ± 5.4 0.381 ± 0.072 < 0.5 ± 0 60.51 ± 1.84 NF3 5 1.5 0.25 134.0 ± 0.6 0.179 ± 0.021 72.0 ± 0 90.67 ± 0.08 NF4 5 1.5 1 142.8 ± 2.2 0.251 ± 0.044 40.0 ± 13.9 68.44 ± 0.56 NF5 10 0.5 0.25 155.2 ± 1.0 0.171 ± 0.019 < 0.5 ± 0 95.03 ± 0.27 NF6 10 0.5 1 166.4 ± 5.5 0.208 ± 0.025 1.3 ± 1.2 81.44 ± 0.41 NF7 10 1.5 0.25 151.8 ± 2.4 0.181 ± 0.016 6.0 ± 0 95.73 ± 0.04 NF8 10 1.5 1 187.2 ± 2.4 0.186 ± 0.006 24.0 ± 0 85.04 ± 0.45 NF9 7.5 1 0.625 154.8 ± 4.7 0.205 ± 0.013 6.0 ± 0 84.93 ± 1.73 A: Initial FIN concentration (mg/ml); B: CLT: FIN (w/w); C: Concentration of PVP solution (% w/v) Table 2 Coded regression equations of response models for optimization of FIN nanosuspension with ANOVA and multiple reliability test ( R 2 ) results. Response Regression equation F value p value R 2 Adjusted R 2 Particle Size (nm) (Y 1 ) = 147.68 + 17.48A + 6.28B + 3.26C + 8.4AC + 7.78BC 128.04 < 0.0001 0.970 0.962 PDI (Y 2 ) = 0.2244-0.0377A-0.0252B + 0.0321C + 0.0222AB-0.0218AC 17.28 < 0.0001 0.812 0.765 Physical Stability (hours) (Y 3 ) = 18.00-10.17A + 17.50B-1.67C-10.33AB + 6.50AC-1.83BC + 6.00ABC 95.23 < 0.0001 0.974 0.964 EE of FIN (%) (Y 4 ) = 83.22 + 6.10A + 1.75B-9.36C-0.6796AB + 3.29AC + 1.13BC 500.23 < 0.0001 0.994 0.992 Note: Factors BC and C in the equation for Y 3 were retained to maintain the hierarchy despite being statistically non-significant. 3.2 Optimization of FIN Dry Powder Formulations As with the optimization of FIN nanosuspension, a 2-level, 3-factor full factorial DoE was used to study the influence and interactions of factors [A: mannitol-nanoparticle ratio (w/w); B: atomization flow rate (L/h); and C: feed rate (ml/min)] on the selected properties of the resulting spray-dried nano-embedded powders [Z 1 : redispersibility (RdI); Z 2 : PDI of the reconstituted nanosuspension; and Z 3 : volumetric size of the spray-dried powder] ( Table S3 ). Mannitol was employed as a carrier in spray drying studies due to its promising safety for pulmonary delivery [ 56 , 57 ]. To preserve the merits of nanoparticles for intranasal delivery, the spray-dried powder should be redispersible to its nanosuspension counterpart, ideally with the same particle size distribution as that freshly prepared from FNP, once in contact with the nasal fluid. Hence, the RdI was set in the range of 0.8–1.2, and the PDI of reconstituted nanosuspension was set as < 0.3. In addition, the particle size of dry powder is a pivotal factor in controlling their nasal deposition profile. Particles less than 5 µm tend to enter the respiratory tract, while particles larger than 20 µm remain in the anterior part of the nose [ 58 , 59 ]. Only powder with a particle size around 10 µm can effectively deposit in the olfactory region [ 60 , 61 ]. The target median particle size of the spray-dry powder was therefore set as 10 µm. The levels of each factor were determined based on previous studies [ 33 , 62 ] and our preliminary data. The physicochemical properties and volumetric particle size distribution of different FIN dry powder formulations obtained from the DoE were presented in Table 3 and Table S6 , respectively. Their RdI varied from 1.02 ± 0.01 to 1.82 ± 0.08, and the PDI of the reconstituted nanosuspensions were close to 0.3. Among different formulations, PF1, PF6, PF7, and PF9 satisfied the pre-set RdI and PDI requirements. The volumetric particle sizes of these dry powder formulations ranged from 4.58 ± 0.57 µm to 10.84 ± 0.47 µm with span 90%. The coded regression equations for each response model of FIN dry powder formulations are listed in Table S7 . The regression equations for redispersibility and particle size had p values 0.90 and agreed with the adjusted R². However, the p- value and R ² values for the PDI regression are 0.715 and 0.200, respectively, indicating that there was no significant factor-response correlation. Hence, the PDI was not included in the numerical optimization process. The Pareto charts of standardized effects of factors and their interactions on the responses are shown in Fig. 4 . All factors and their interactions, except for AC and ABC, showed a positive influence on the RdI of the dry powder. When factor A (mannitol-nanoparticle ratio) increased, the total solute concentration and drying time of nanosuspension droplets would also increase, leading to adverse outcomes on the nanoparticle stability and RdI. The most significant factor affecting RdI was factor B (atomization flow rate) in this study. This was not surprising and could be ascribed to greater shear stresses exerted on the nanoparticle surface during feed atomization. Moreover, a rise in factor C (feed rate) would generate larger droplets, resulting in a longer drying time and stronger thermal stress on the nanoparticles, hence the corresponding increase in RdI. The interaction terms AB and BC also shared the same trend and effect on RdI with their individual factors. Interestingly, all factors and their interactions did not have a significant effect on the PDI of the reconstituted nanosuspension. This implied that these factors had a minimal impact on the uniformity of nanoparticles embedded in the resulting spray-dried powders. Regarding the particle size of the powder, both factors A and B and their interactions AB and BC displayed a negative effect. An increase in factor B would yield smaller droplets and, thus, smaller particles. However, the trend for factor A deviated from literature reports, where particle size increases as solute concentration in feed solution increases [ 63 ]. We speculate that the denser dry powders (PF3 and PF4) produced by high solute concentrations with low atomization flow rates were fractured during the dispersion from the nasal device, resulting in a smaller volumetric particle size. The regression models for redispersibility and particle size were simplified by removing non-significant terms (Table 4 ), and the desirability function was applied to optimize the FIN nano-embedded powder formulation. A moderate level of drug loading can reduce medication dosage. Since an increase in factor A (mannitol-nanoparticle ratio) is correlated with increasing RdI, and it is preferable to minimize the excipients used, the level of A is set as the lowest in the design space. The highest desirability value was achieved when A = 4:1 ( w/w ), B = 407 (L/h), and C = 4.2 (mL/min), and the corresponding predicted RdI and volumetric median particle size of the dry powder were 1.1 and 10 µm, respectively. Model validation was performed for fabricating the FIN dry powder ( n = 3) using the optimized parameters. The obtained dry powder had a RdI of 1.09 ± 0.04 and a median particle size of 11.31 ± 1.7 µm, congruent with the predicted properties. The PDI (0.272 ± 0.019) of the nanosuspension reconstituted from the dry powders also fulfilled the target requirement. As shown in Fig. 5 a, the optimized dry powder could be reconstituted back to nanosuspension without significant changes in particle size distribution compared to the freshly prepared nanosuspension. The volumetric size of the dry powder was 11.31 ± 1.7 µm, which was expected to be effectively deposited in the olfactory region of the nasal cavity. The drug contents of FIN (1.55 ± 0.09%) and CUR (0.68 ± 0.11%) of the dry powder were consistent with the theoretical values (i.e., 1.6% for FIN and 0.8% for CUR). Table 3 Physicochemical properties of FIN dry powders prepared based on the 3-factor 2-level factorial design (n = 3). Powder Formulation (PF) Factors with levels Responses and results A B C RdI(Z 1 ) PDI (Z 2 ) Particle Size D 50 (µm) (Z 3 ) PF1 10 601 1.5 1.05 ± 0.03 0.299 ± 0.044 8.69 ± 0.21 PF2 10 601 4.5 1.02 ± 0.01 0.347 ± 0.092 9.91 ± 0.28 PF3 10 357 1.5 1.60 ± 0.08 0.338 ± 0.061 5.16 ± 0.62 PF4 10 357 4.5 1.82 ± 0.08 0.310 ± 0.020 4.58 ± 0.57 PF5 4 357 1.5 1.14 ± 0.02 0.303 ± 0.038 8.95 ± 1.14 PF6 4 357 4.5 1.08 ± 0.05 0.293 ± 0.038 10.84 ± 0.47 PF7 4 601 1.5 1.04 ± 0.06 0.285 ± 0.030 8.34 ± 0.28 PF8 4 601 4.5 1.30 ± 0.04 0.305 ± 0.029 7.44 ± 0.49 PF9 7 473 3 1.13 ± 0.03 0.298 ± 0.020 8.38 ± 0.53 A: Mannitol: nano (w/w); B: Atomization gas flow rate (L/h); C: Feed rate (ml/min) Table 4 Coded regression equations of response models for optimization of FIN dry powders with ANOVA and multiple reliability test ( R 2 ) results. Response Regression equation F value p value R 2 Adjusted R 2 Redispersibility (Z 1 ) = 1.26 + 0.1150A + 0.1850B + 0.0475C + 0.1558AB + 0.0717BC 170.76 < 0.0001 0.977 0.971 Volumetric Particle Size of dry powder (µm) (Z 3 ) = 7.99-0.9046A-1.61B + 0.2029C-0.6063AB-0.5738BC 62.77 < 0.0001 0.940 0.925 Note: Factor C in the equation for Z 3 was retained to maintain the hierarchy despite being statistically non-significant. 3.3 Characterization of the FIN Nano-Embedded Dry Powder Formulation Presented in Fig. 5 b and Fig. 5 c are the SEM images of the optimized FIN dry powders. The particles exhibited a spherical morphology with embedded nanoparticles (as indicated by the black frame), contributing to their good flowability for ease of powder loading in nasal spraying devices. Considering that a higher aspect ratio of particles could augment powder deposition in the alveolar region, the spherical morphology minimizes the fraction of particles inadvertently deposited in deep lung areas during intranasal application [ 64 ]. As seen in the DSC and PXRD profiles ( Figs. S2a and S2b ), except PVP, all other raw materials, their physical mixture counterpart, and the optimized FIN dry powder were crystalline in nature. The TGA analysis revealed that approximately 3.01% of residual moisture was present in the optimized dry powders, and its moisture content gave a temperature-dependent reduction to 0.91–1.40% upon 1 month of storage ( Fig. S2 c ). This was not surprising as unbound water in the powder evaporated with a moisture balance. The optimized FIN dry powder possessed satisfactory stability as no significant phase transformation and reduction in drug assays were detected under various storage conditions. Presented in Fig. S2 d are the FTIR spectra of different samples. A very broad peak can be seen from around 3,600 cm − 1 to 3,100 cm − 1 in the optimized FIN dry powder. This is likely due to the formation of intermolecular hydrogen bonding between CUR and PVP [ 65 ], which improved the stability of the nanoparticles. Regarding the in vitro drug release, while the optimized FIN dry powder showed rapid and good redispersion (RdI < 1.2) in simulated nasal fluid, neither FIN nor CUR were detectable by HPLC throughout the 3-hour experiment. The FIN or CUR in the physical mixture in the simulated nasal fluid also could not be detected. However, the release of FIN and CUR from the optimized FIN dry powder surpassed those of its physical mixture counterpart in an 80:20 ( v/v ) solution of simulated nasal fluid and ethanol ( Fig. S3 ). It is believed that no drug release occurred in the physical mixture due to its limited solubility, but for the nanosuspension reconstituted from the optimized FIN dry powder, most of the drugs were well encapsulated and protected within the nanoparticles. The effective encapsulation of drugs within the nanoparticles also renders the optimized dry powder less susceptible to nasal mucociliary clearance (typically occurring every 10–20 minutes) [ 66 ], making it suitable for nose-to-brain drug delivery. The nasal deposition studies of the optimized FIN dry powder were conducted using two different nasal anatomical models at varying flow rates of 0, 7.5, and 15 L/min. The flow rates were selected based on the typical breathing patterns pertinent to clinical applications: a flow rate of 15 L/min mimics normal steady breathing [ 67 ], 7.5 L/min corresponds to slow inhalation [ 39 ], and 0 L/min represents an absence of breathing or a breath-holding scenario [ 14 , 68 ]. All nasal models were connected to an NGI to simultaneously study powder deposition in the nose and the respiratory tract. The powders demonstrated excellent dispersibility upon actuation of the nasal device, with the nasal device fraction consistently below 5% (Fig. 6 ), except for the 3D-printed nasal cast at 0 L/min flow rate. These findings suggest that the nasal device is generally effective for administering powder formulations to the nasal cavity. The high deposition in the nasal device (~ 20%) observed for the 3D-printed nasal cast at 0 L/min flow rate may be attributed to the powder backflow from the respiratory region of the cast to the exterior of the nasal device due to insufficient energy from the flow. The olfactory pathway is an important route for nose-to-brain delivery [ 69 , 70 ]. In the 3D-printed nasal cast (Fig. 6 a), the deposition fractions of optimized FIN dry powders in the olfactory region were 45.4% for 15 L/min, 45.2% for 7.5 L/min, and 48.5% for 0 L/min. Hence, as high as 45% of the optimized FIN dry powder can deposit in the olfactory region. In the AINI model (Fig. 6 b), the deposition fractions of optimized FIN dry powder in the olfactory region were 8.6% for 15 L/min, 4.7% for 7.5 L/min, and 7.3% for 0 L/min. Aside from the olfactory region being the primary focus for nose-to-brain delivery, the highly vascularized respiratory region also warrants attention, as drugs can be absorbed in this area for systemic delivery. In addition, parts of the turbinate region innervated by the trigeminal nerve can transport the drug from the nose to the brain via the trigeminal pathway [ 5 , 68 ]. In the 3D-printed nasal cast, the deposition fractions of optimized FIN dry powder in the respiratory region were 48.3% for 15 L/min, 48.5% for 7.5 L/min, and 29.2% for 0 L/min. In the AINI model, the deposition fractions of optimized FIN dry powder in the respiratory region (sum of nasopharynx, turbinates, and vestibule) were 62.3% for 15 L/min, 62.3% for 7.5 L/min, and 85.0% for 0 L/min. In detail, the deposition fractions of optimized FIN dry powder in the turbinate region were 25.6% for 15 L/min, 29.2% for 7.5 L/min, and 62.5% for 0 L/min. The discrepancy in regional deposition profiles between the 3D-printed nasal cast and the AINI was not surprising and can be ascribed to the differences in the design and construction materials of these models. Since the olfactory region of the 3D-printed nasal cast has a larger surface area, the deposition fraction was higher in this model. Apart from the design, the 3D-printed nasal cast was made of polylactic acid, while the AINI model was fabricated from stainless steel. The adhesion capabilities, texture, and potential impact of surface charges on dry powders may exhibit variations. In the present study, the FIN nano-embedded powders displayed a greater propensity to adhere to plastic surfaces. Powders in the olfactory region of the AINI model may have unintentionally fallen into the respiratory region (especially during the disassembly of the AINI), resulting in a lower deposition fraction. The in vitro - in vivo correlation of drug deposition using these nasal models should be further investigated. It is worth noting that the inspiratory flow rate may vary among patients of different ages and disease states. Therefore, a holistic evaluation of how the inspiratory flow rate may influence the nasal deposition is desired. Perkušić et al. found that an increased inspiratory flow rate from 0 L/min to 60 L/min was associated with reduced drug deposition in the olfactory region [ 14 ], while Rigaut et al. found that flow rate (0, 15, and 60 L/min) was not a significant influencing factor for olfactory deposition [ 67 ]. In this study, the deposition fraction of the olfactory region was independent of the flow rate for the optimized FIN powder formulation under different flow rates in two different nasal models ( Fig. S4 ). The reason for this observation could be due to the restricted range of inspiratory flow rate (0–15 L/min) studied. Besides, the manufacturer of the Unidose Nasal Spray Device (Aptar Pharma) has suggested that simultaneous actuation of the nasal device and inspiration is not required because the nasal device can generate sufficient air pressure for powder dispersion [ 67 ]. Hence, the optimized powder formulation is suitable for patients with different inspiratory flow rates. 3.4 In Vitro Cytotoxicity Studies of the Optimized FIN Nano-embedded Dry Powder Formulation The cytotoxicity of FIN nanosuspension reconstituted from the optimized dry powder was evaluated in various cell lines. SH-SY5Y and PC 12 cells were used as neuronal cell models, while RPMI 2650 and Calu-3 were used as nasal epithelial cell models [ 71 ]. In addition, the cytotoxicity of raw FIN and a physical mixture of the optimized powder formulation composition were also investigated for comparison purposes. The concentration range studied (2.5–1,000 nM) was chosen based on literature reports and clinical pharmacokinetics of FIN [ 72 , 73 ]. As presented in Fig. 7 , the cell viability for all groups was around 100%, with no significant difference observed. This result indicates that the formulation has an acceptable in vitro safety profile. 3.5 In Vivo Neuroprotective Effects of the Optimized FIN Nano-embedded Dry Powder Formulation A schematic diagram of the study design of in vivo neuroprotective effects is depicted in Fig. 8 a. FIN nanosuspension was reconstituted from the optimized dry powder and separately delivered to the mice with MCAO surgery via intravenous (IV) and intranasal (IN) administration. To assess the neuroprotective effect with greater clinical relevance, the tested drugs were administrated 30 minutes after stroke surgery. As shown in Fig. 8 b, the brain slices were stained with Nissl solutions on day 2 and the infarct regions were marked by the red line. The infarct size was significantly reduced to 1.01 ± 0.12 mm 2 in the IV-treated group compared with the untreated MCAO group (1.54 ± 0.10 mm 3 ). Remarkably, the infarct size was further reduced to 0.71 ± 0.07 mm 2 in the IN-treated group (Fig. 8 c ) . To precisely measure the cerebral infarct size, we also employed the 9.4T animal magnetic resonance imaging system (MRI) 24 hours post-treatment. Figure 9 a shows the mouse brain MRI images with clear cerebral infarction after MCAO surgery in the left hemispheres, with an average infarct volume of 2.93 ± 0.71 mm 3 . The IV-treated mice had a lower infarct volume of 2.13 ± 0.85 mm 3 , while the IN-treated group gave a significant reduction to 1.31 ± 0.57 mm 3 (Figs. 9 b and 9 c). The neuroprotective effects were also evaluated using the adhesive removal test, balance beam test, rotarod test, and the Longa score. As expected, the time required for the mice to remove the adhesive tapes from their bilateral paws after MCAO surgery was significantly increased due to the neurological deficit after acute ischemia imposed by the MCAO surgery (Fig. 10 a, b). However, the time required for MCAO mice with IV and IN treatment to remove the adhesive tapes decreased significantly compared to untreated MCAO mice on day 2 and day 3 (Figs. 10 a and 10 b) after stroke surgery. Notably, the effect on the IN-treated group on day 3 was more significant than that of the IV-treated group, as evidenced by the smaller p -value. Likewise, MCAO mice treated with reconstituted FIN nanosuspension intranasally required less average time to cross the beam compared to untreated MCAO mice and IV-treated MCAO mice (Figs. 10 c and 10 d ) . For the rotarod test, however, the time spent on the rod for treated groups was not statistically significant from that in the untreated group on day 2 (Fig. 10 e), possibly due to the sensitivity of the rotarod test for the stroke model [ 74 ]. Nevertheless, a statistically significant difference (p < 0.01) was observed between the MCAO + IN FIN and MCAO groups on day 3 (Fig. 10 f). The Longa scores of mice in both treatment groups significantly decreased compared to the untreated MCAO group (Fig. 10 g). Taken together, these results suggest that the IN administration outperformed IV administration in terms of alleviating neurological deficits after a stroke. Furthermore, a western blot analysis was performed on post-stroke mouse brains to understand the neuroprotective mechanism. Expression of pro-apoptotic proteins BAX and CC3, and the anti-apoptotic protein BCL-2 in the peri-infarct tissue of our MCAO stroke model were determined [ 49 ]. This was due to reports that FIN could increase the expression of BCL-2 and decrease the expressions of CC3 and BAX [ 75 , 76 ]. As shown in Fig. 11 , administration of the reconstituted FIN dry powder for nanosuspension by IV and IN routes showed significant neuroprotective effects by reducing the expression of apoptosis proteins CC3 and BAX and increasing the expression of anti-apoptotic protein BCL-2. Of note, expression of BAX in the peri-infarct tissue was statistically significantly lower in IN-treated mice compared to that in IV-treated mice. BCL-2 expression was also numerically higher in IN-treated mice relative to IV-treated mice despite the lack of statistically significant differences. Nevertheless, these results highlight that IN delivery of FIN nanoparticles was more effective in reducing infarct size in the peri-infarct tissue than IV delivery. It is noteworthy that the neuroprotective effects could be achieved after single-dose IN administration. The non-invasive and convenient IN administration method also presents a unique treatment modality where FIN nanoparticles (in form of dry powder or reconstituted nanosuspension) can be timely administered by ambulance staff or caregivers to patients experiencing acute ischemic stroke symptoms before hospital admission. This strategy can attenuate neuronal injury in the hyperacute phase of ischemic stroke between stroke onset and in-hospital treatment procedures and promote post-stroke functional recovery. Whilst cardiovascular adverse effects such as bradycardia and atrioventricular blockade have been observed in patients on chronic oral FIN treatment for multiple sclerosis [ 77 ], we do not anticipate significant safety issues arising from single-dose IN nanoparticle administration due to its excellent cytocompatibility after single-dose treatment. A constraint of the current study is that mice were administered IN with reconstituted FIN nanosuspensions instead of FIN nano-embedded powder as precise powder administration to the small nares of rodents is challenging. Nonetheless, this study provides proof-of-concept evidence of the neuroprotective effects by FIN nanoparticles. It is anticipated that the administration of nano-embedded powders would offer prolonged nasal retention and thus further enhance nose-to-brain FIN transport and neuroprotective efficacy. Studies are ongoing to devise an appropriate technique for administering powder into the nares of rodents and investigate the dose-response relationship and pharmacokinetics of intranasally administered FIN nano-embedded powder in rodent MCAO models. The ultimate goal is to identify an optimal powder dose that can serve as an effective and rapid neuroprotective therapy after an acute ischemic stroke. 4. Conclusions A FIN nano-embedded dry powder formulation for intranasal application was developed using a full factorial design of experiments. The optimized FIN nanosuspension had a particle size of 134.0 ± 0.6 nm, a satisfactory PDI, and acceptable stability. The nanosuspension was then spray-dried into a nano-embedded microparticles dry powder with the aid of mannitol. The optimized dry powder exhibited excellent redispersibility (RdI = 1.09 ± 0.04) and good drug deposition in the olfactory region. The deposition fractions in the olfactory region were found to be independent of the nasal inspiratory flow rate, rendering it suitable for patients with different clinical conditions. It also had acceptable safety profiles in both nasal and brain cell models. Improved behavioral test results, reduced infarct volume, altered expressions of anti-apoptotic and pro-apoptosis proteins were observed following IN administration of the reconstituted FIN nanosuspension in a MCAO mouse model. This study demonstrates the neuroprotective effects of IN and IV FIN nanoparticles, with IN administration showing superiority for ischemic stroke management. Investigations into dose-response effects and pharmacokinetics of the FIN nano-embedded dry powder via IN and IV administrations are ongoing to further optimize its efficacy. Abbreviations AINI Alberta Idealised Nasal Inlet ATCC American Type Cultural Collection BAX B-cell lymphoma 2 associated X BCL-2 B-cell lymphoma 2 CC3 cleaved Caspase-3 CLT cholesterol CQA critical quality attributes CUR curcumin DL drug loading DLS dynamic light scattering DMEM/F12 Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 DMEM Dulbecco’s Modified Eagle’s Medium DSC differential scanning calorimetry DoE Design of Experiment EE encapsulation efficiency EtOH ethanol F-12K Kaighn's Modification of Ham's F-12 FBS fetal bovine serum FIN fingolimod FTIR Fourier-transform infrared spectroscopy IN intranasal IV intravenous MCAO middle cerebral artery occlusion MEM Minimum Essential Medium MeOH methanol MIVM multi-inlet vortex mixer MRI magnetic resonance imaging NGI next generation impactor PBS phosphate-buffered saline PDI polydispersity index PVP Polyvinylpyrrolidone PXRD powder x-ray diffractometry RdI redispersibility index Re Reynold number S.D. standard deviation TEM transmission electron microscopy TFA trifiuoroacetic acid TGA thermogravimetric analysis UPW ultra-purified water Declarations Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethics approval All animal studies followed the ethical policies and guidelines recommended by ARRIVE (Animal Research: Reporting of In Vivo Experiments) and National Research Council's Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of the Animal Experimentation of Jinan University (Approval No.: 20230901-0012). Funding This research was financially supported by the University of Hong Kong (Project Number: 104006626), the Health@InnoHK programme from the Innovation and Technology Commission, Hong Kong SAR government, the Guangdong Basic and Applied Basic Research Foundation, China (2023B1515120035, 2024A1515012035) and the Science and Technology Planning Project of Guangdong Province, China (2020A0505100045). Author Contributions Xinyue Zhang : Conceptualization, Methodology, Investigation, Formal Analysis, Writing – Original Draft, Visualization. Guangpu Su : Methodology, Investigation, Formal Analysis. 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Statements & Declarations Supplementary Files GA.png Graphical Abstract Supplementarymaterialfinalized.docx VideosS1.mov Cite Share Download PDF Status: Published Journal Publication published 01 Nov, 2024 Read the published version in Drug Delivery and Translational Research → Version 1 posted Editorial decision: Major Revisions Needed 06 Sep, 2024 Reviewers agreed at journal 28 Jul, 2024 Reviewers invited by journal 22 Jul, 2024 Editor assigned by journal 11 Jul, 2024 First submitted to journal 09 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4715108","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":330372321,"identity":"1af1bfc7-97a2-41c4-ba1c-9075cdae4899","order_by":0,"name":"Xinyue Zhang","email":"","orcid":"","institution":"The University of Hong Kong Department of Pharmacology and Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Xinyue","middleName":"","lastName":"Zhang","suffix":""},{"id":330372322,"identity":"a81885c2-c88b-48ac-852b-19917b4ab459","order_by":1,"name":"Guangpu Su","email":"","orcid":"","institution":"Jinan University First Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Guangpu","middleName":"","lastName":"Su","suffix":""},{"id":330372323,"identity":"0c90b842-73cf-41e7-bf80-edc2138daf46","order_by":2,"name":"Zitong Shao","email":"","orcid":"","institution":"University of Hong Kong Li Ka Shing Faculty of Medicine Department of Pharmacology and Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Zitong","middleName":"","lastName":"Shao","suffix":""},{"id":330372324,"identity":"4841c49f-83f6-4b3d-abe9-3c679eaf2f89","order_by":3,"name":"Ho Wan Chan","email":"","orcid":"","institution":"The University of Hong Kong Department of Pharmacology and Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Ho","middleName":"Wan","lastName":"Chan","suffix":""},{"id":330372325,"identity":"f51f3e7e-eb92-4532-9583-8d6518b7fbfd","order_by":4,"name":"Si Li","email":"","orcid":"","institution":"The University of Hong Kong Department of Pharmacology and Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Si","middleName":"","lastName":"Li","suffix":""},{"id":330372326,"identity":"52eaeac0-9d56-47c3-86c2-c2fb50737adb","order_by":5,"name":"Stephanie Chow","email":"","orcid":"","institution":"The University of Hong Kong Department of Pharmacology and Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Stephanie","middleName":"","lastName":"Chow","suffix":""},{"id":330372327,"identity":"79a505d4-c44e-413b-9014-f2f78899af91","order_by":6,"name":"Chi Kwan Tsang","email":"","orcid":"","institution":"Jinan University First Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chi","middleName":"Kwan","lastName":"Tsang","suffix":""},{"id":330372328,"identity":"11d94b63-17c7-4f21-96c2-90b8022c8517","order_by":7,"name":"Shing Fung Chow","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArklEQVRIiWNgGAWjYFACxgfMQFLOgIeBgZnhAFFamA1AWoxJ15K4gWgt/P2HGT8XVBxO385zxoC54AwRWiRuJDNLzzhzOHdnb48B84wbxLjrBv8xZt6227kbzvMYMPN8IEKH/PnDbCAt6QZEazE4kAzWkmBwFugwHmIcZgjyC8+Z/4YbzhwrOMxDjPflzgNDjKciTd7gTPLGxzzHiNCCAg6QqmEUjIJRMApGAQ4AAPGRNkgyuUVxAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-4588-5346","institution":"The University of Hong Kong Department of Pharmacology and Pharmacy","correspondingAuthor":true,"prefix":"","firstName":"Shing","middleName":"Fung","lastName":"Chow","suffix":""}],"badges":[],"createdAt":"2024-07-10 02:53:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4715108/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4715108/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13346-024-01721-8","type":"published","date":"2024-11-01T16:20:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62755244,"identity":"b2c5d09a-1fbb-48c5-bb7e-72c320765896","added_by":"auto","created_at":"2024-08-19 06:29:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":253426,"visible":true,"origin":"","legend":"\u003cp\u003eThe experimental setup for the preparation of FIN nanosuspensions.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4715108/v1/a88ef4b284a4a7249df8640c.png"},{"id":62755253,"identity":"1288f2fa-1f65-4a60-8f35-8555b409bd44","added_by":"auto","created_at":"2024-08-19 06:29:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":346500,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic presentation of (a) the 3D printed nasal cast; (b) the AINI coupled with the NGI for the aerosol deposition assessment.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4715108/v1/38fb177f92c733784b87f20b.png"},{"id":62757148,"identity":"779745ed-b9d4-4b55-b74a-ecf5a7b6b0bd","added_by":"auto","created_at":"2024-08-19 06:53:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":136563,"visible":true,"origin":"","legend":"\u003cp\u003ePareto charts of standardized effects of factors and their interactions on (a) particle size of the FIN nanosuspension, (b) PDI of the FIN nanosuspension, (c) stability of the FIN nanosuspension and (d) EE of FIN nanosuspension. Factors and interactions that cross the horizontal black line show statistically significant effects on the responses. A blue bar stands for negative effects on the responses, while orange bar stands for positive effects on the responses\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4715108/v1/a5961fd5f3391e6b5a4052d1.png"},{"id":62756655,"identity":"35ad6ac8-3ec2-4987-a7b8-27a200924948","added_by":"auto","created_at":"2024-08-19 06:45:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":99393,"visible":true,"origin":"","legend":"\u003cp\u003ePareto charts of standardized effects of factors and their interactions on (a) redispersibility of the FIN dry powder, (b) PDI of the reconstituted nanosuspension and (c) volumetric particle size of the FIN dry powder. Factors and interactions that cross the horizontal black line show statistically significant effects on the responses. Blue bar stands for negative effects on the responses, while orange bar stands for positive effects on the responses.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4715108/v1/ccf716bba18e64c195ff19ad.png"},{"id":62756038,"identity":"32c1150c-ae40-4a26-83e3-f11a4b25ea8d","added_by":"auto","created_at":"2024-08-19 06:37:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":938298,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Differential intensity with size distribution of the optimized FIN nanosuspension and the reconstituted FIN dry powder for nanosuspension. Scanning electron microscopy (SEM) images of optimized FIN dry powder at (b) × 700 magnification (scale bar = 50.0 μm) and (c) ×18.0 k magnification (scale bar = 3.0 μm). The embedded nanoparticles were shown in the black frame.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4715108/v1/5e8e89e1d58a65a372dea3fd.png"},{"id":62755250,"identity":"3a54c94b-a507-4eb6-b27f-066f29f79767","added_by":"auto","created_at":"2024-08-19 06:29:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":70925,"visible":true,"origin":"","legend":"\u003cp\u003eFraction of recovered dose on nasal model regions, nasal device and NGI stages of optimized FIN dry powder at inspiratory flow rates of 0, 7.5 and 15 L/min using (a) the 3D-printed nasal cast and (b) the AINI model.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4715108/v1/a75c2a97da61b3dbc5bd3f9b.png"},{"id":62755249,"identity":"6790b9f9-3a2b-4c08-91ab-5e9453a1c0ce","added_by":"auto","created_at":"2024-08-19 06:29:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":77126,"visible":true,"origin":"","legend":"\u003cp\u003eThe cytotoxicity of raw FIN, the physical mixture of the formulation components, and reconstituted FIN dry powder for nanosuspension in (a) Calu-3 cells, (b) RPMI 2650 cells, (c) SH-SY5Y cells, and (d) PC 12 cells at the FIN concentration ranged 2.5-1000 nM.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4715108/v1/a079c35e5aedd30e8911d0fb.png"},{"id":62756039,"identity":"505a946d-b785-4432-9606-21d26850d667","added_by":"auto","created_at":"2024-08-19 06:37:51","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":901519,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Experimental scheme of the \u003cem\u003ein vivo\u003c/em\u003e animal experiments. (b) Representative images of the brain slices stained by Nissl staining solution on day 2. The infarct regions were marked by red line. (c) The infarct volumes of brain in each group (n=3). Data are presented as means ± SD, * p\u0026lt;0.05, ** p\u0026lt;0.01, **** p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4715108/v1/0590b1460cb2f59318ce461d.png"},{"id":62755245,"identity":"1707a0f3-9ae0-46fa-a847-0d0edf861024","added_by":"auto","created_at":"2024-08-19 06:29:50","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":670785,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Representative images of brain MRI in each group. The infarct region is circled by red line. (b) Representative images of the infarct core (upper left) and three-dimensional images of brain infarction (upper right). The coronal section, horizontal section, and sagittal section are shown from bottom left to right in MCAO mice treated with 0.9% saline intranasally, reconstituted FIN dry powder for nanosuspension intravenously, and reconstituted FIN dry powders for nanosuspension intranasally on day 2. (c) The infarct volumes of brain in each group (n=6). Data are presented as means ± SD, ** p\u0026lt;0.01, **** p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4715108/v1/1fc868ba33ecb1fe9a2f6669.png"},{"id":62755252,"identity":"2bc3e394-3ca6-4102-afb1-dce0700659f7","added_by":"auto","created_at":"2024-08-19 06:29:51","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":134280,"visible":true,"origin":"","legend":"\u003cp\u003eThe neurological functional assessments of mice. Adhesive removal tests on day 2 (a) and day 3 (b), Cross beam tests on day 2 (c) and day 3 (d), Rotarod tests on day 2 (e) and day 3 (f), Neurological deficit evaluation by Longa scores on day 3 (g). Data are presented as means ± SD, n=8 for day 2 and n=7 for day 3. * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001 and **** p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-4715108/v1/6e0df99167f6b26129cd06cd.png"},{"id":62756040,"identity":"549bf254-684b-448e-ab8b-ef6c9b37d184","added_by":"auto","created_at":"2024-08-19 06:37:51","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":309712,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Representative western blot images showing the protein expression of BAX, CC3 and BCL-2 in each group after treatment on day 3. Quantification of the Western blot results showing the relative expression of (b) BAX (c) CC3 and (d) BCL-2 to β-tubulin in each group. Data are presented as means ± SD, n = 6, * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-4715108/v1/cc32b34c9b2e482de33af546.png"},{"id":68207264,"identity":"221e97f9-fe92-400a-a4d9-97cce0e215e2","added_by":"auto","created_at":"2024-11-04 16:36:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5784954,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4715108/v1/96374092-3d01-4082-a160-1cf4511ef570.pdf"},{"id":62756035,"identity":"279e9e9c-a0a2-4b02-84fc-61a52f069501","added_by":"auto","created_at":"2024-08-19 06:37:50","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":656903,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-4715108/v1/4de76e905d805f73bce1f1d5.png"},{"id":62755255,"identity":"8a06a33e-c083-4cb3-b0ce-06c9c796f3d7","added_by":"auto","created_at":"2024-08-19 06:29:51","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5990083,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterialfinalized.docx","url":"https://assets-eu.researchsquare.com/files/rs-4715108/v1/88ede29fef891598cc575234.docx"},{"id":62755258,"identity":"57e80769-1285-432f-b515-c79756b109c4","added_by":"auto","created_at":"2024-08-19 06:29:52","extension":"mov","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":123354536,"visible":true,"origin":"","legend":"","description":"","filename":"VideosS1.mov","url":"https://assets-eu.researchsquare.com/files/rs-4715108/v1/9f1096d72b723813f4223e5a.mov"}],"financialInterests":"","formattedTitle":"Rational Development of Fingolimod Nano-embedded Microparticles as Nose-to-Brain Neuroprotective Therapy for Ischemic Stroke","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eStroke is a leading cause of permanent disability worldwide, affecting approximately 15\u0026nbsp;million global citizens annually [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite ongoing improvements in stroke care and rehabilitation, many patients still suffer from different degrees of lifelong disabilities. Stroke accounts for an estimated 3\u0026ndash;4% of total medical expenses in Western countries [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], not only affecting patients\u0026rsquo; health and quality of life but also imposing a considerable long-term financial burden on healthcare systems. Ischemic stroke is the most prevalent form of stroke and is responsible for around 85% of stroke cases [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Although intravenous thrombolysis and mechanical thrombectomy are clinically available for acute ischemic stroke treatment, only a small portion of patients benefit due to a restricted therapeutic time window, resulting in a high rate of disability among stroke patients [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Consequently, there is a need to develop rapid and effective neuroprotective therapies for the management of acute ischemic stroke.\u003c/p\u003e \u003cp\u003eUpon the onset of ischemic stroke, blood supply to the neurons is interrupted immediately, resulting in substantial cell death [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Timely treatment has a significant impact on relieving the severity of the patient's disease condition and their degrees of lifelong disabilities. However, it typically takes several hours for oral neuroprotective medications to reach their optimal treatment concentration in the brain. Hence, alternative drug administration methods with faster onset are required, especially for patients who are unable to take medicines orally during an acute ischemic stroke episode. Nose-to-brain drug administration has become attractive nowadays because of its minimal invasiveness, easier self-administration, higher effectiveness with less systemic side effects, and more direct route to the central nervous system [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Previous research has indicated the drug can be delivered to the brain rapidly after intranasal administration [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Integrating intranasal administration with nanotechnology further enhances the potential of this delivery approach, as it prolongs drug residence time at the absorption site, increases cellular internalization, and regulates the release of encapsulated drugs [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Previous studies have also demonstrated that intranasal delivery of drug nanoparticles could achieve improved bioavailability [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and high brain targetability [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] while concurrently reducing off-target concentration in the bloodstream [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Moreover, drug encapsulation within nanoparticles can minimize degradation risks [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and promote rapid brain uptake [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The above-mentioned properties are highly sought after for the intranasal administration of neuroprotective nanoparticles, especially for targeted transport to the ischemic brain.\u003c/p\u003e \u003cp\u003eCompared with nasal sprays, nasal powders possess unique benefits such as extended retention time and superior stability against enzymatic degradation within the nasal cavity [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Furthermore, nasal powders can be reconstituted in an aqueous buffer as a nasal solution or suspension to offer more flexible dosing according to specific clinical needs [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. A notable obstacle in the engineering of nanoparticle-loaded powders is the maintenance of appropriate redispersibility of dry powder back into the nanoparticle once contact with the nasal fluid to preserve the therapeutic merits of nanoparticles. Another major challenge in fabricating dry powders for optimal nose-to-brain delivery lies in particle size control, as only particles with a diameter of around 10 \u0026micro;m can achieve greater deposition in the olfactory region, i.e., the primary target believed to be responsible for nose-to-brain delivery [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In the present study, spray drying was selected for converting nanosuspensions into dry powders owing to its commercial availability for simplified scale-up, as well as its ability to tailor particles for targeted intranasal delivery to the olfactory region [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFingolimod (FIN) is an oral drug approved for the treatment of multiple sclerosis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It has also demonstrated neuroprotective effects in various animal studies and clinical trials. For instance, ischemic stroke models of mice that received FIN intravenously or intraperitoneally showed a decrease in infarct size and improved behavioral testing results [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Moreover, stroke patients treated with standard management and oral FIN medication beyond the 4.5 h treatment window for intravenous thrombolysis (tissue plasminogen activator) displayed reduced secondary tissue injury, decreased neurological impairments, and improved post-stroke recovery compared to patients who received standard management alone [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Consequently, FIN is a promising neuroprotective drug with therapeutic potential for acute ischemic stroke. Curcumin (CUR), a polyphenol found in turmeric, has also shown neuroprotective effects in both hemorrhagic and ischemic stroke cases [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, the delivery of FIN and CUR to the brain still requires optimization for improved clinical outcomes.\u003c/p\u003e \u003cp\u003eWhile separate studies have shown the therapeutic potential of FIN for acute ischemic stroke, no research has yet studied its neuroprotective effect through intranasal nanotherapy. This delivery strategy confers the unique advantage of rapid treatment for patients during ambulance transportation, obviating the requirement for conscious patient cooperation. Hence, the objective of this study was to develop FIN nano-embedded nasal powders for rapid neuroprotection after the onset of acute ischemic stroke. To this end, a full factorial design of experiments was conducted to examine the effects of and optimize critical formulation and processing parameters for FIN nanosuspension and its nano-embedded dry powder formulations. The optimized powder formulation was characterized for various pharmaceutical properties, such as aqueous redispersibility, particle size, nasal deposition profile, crystallinity, cytotoxicity, and stability. Lastly, the neuroprotective effects of nasally administered FIN nanoparticles were evaluated through neurological functional tests and infarct size measurements in a well-established acute ischemic stroke model.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eFingolimod (FIN, \u0026gt; 99.9% purity) was purchased from Hefei Hirisun Phamatech (Hefei, China). Curcumin (CUR, \u0026gt; 99.5% purity) was sourced from Yung-Zip Chemicals (Taichung, Taiwan). Methanol (MeOH) and ethanol (EtOH) were obtained from VWR BDH Chemicals (VWR International S.A.S., Fontenay-sous-Bois, France). Ultra-purified water (UPW) was generated using a Barnstead NANOpure Diamond system (Thermo Fisher Scientific, Waltham, MA, USA). Polyvinylpyrrolidone (PVP K30) and cholesterol (CLT) were procured from Sigma-Aldrich (St. Louis, MO, USA), and Mannitol (Pearlitol 160C) was purchased from Roquette (Lestrem, France). Trifluoroacetic acid (TFA), Dulbecco\u0026rsquo;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12), Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM), Minimum Essential Medium (MEM), Kaighn's Modification of Ham's F-12 Medium (F-12K medium), 0.25% (w/v) trypsin-EDTA, phosphate-buffered saline (PBS, 10\u0026times;), fetal bovine serum (FBS), and antibiotic\u0026ndash;antimycotic (100\u0026times;) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Additionally, a variety of cell lines were obtained from the American Type Cultural Collection (ATCC; Manassas, VA, USA), including PC 12 cells, RPMI 2650 cells, Calu-3 cells, and SH-SY5Y cells. Nissl Staining Solution (Cat. No. C0117) was purchased from Shanghai Beyotime Biotechnology Co., Ltd (Shanghai, China). Polyvinylidene difluoride membrane was purchased from Bio-Rad (California, USA). Primary antibodies against Cleaved Caspase-3 (CC3) (Cat. No. 9664) and β-Tubulin proteins (Cat. No. #2146S) were purchased from Cell Signaling Technology (Massachusetts, USA) whose t against B-cell lymphoma 2 (BCL-2) (Cat. No. WL01556) and BCL-2 associated X (BAX) (Cat. No. WL01637) proteins were purchased from Wanlei Bio (Liaoning, China). Horseradish peroxidase-labeled goat anti-rabbit IgG(H\u0026thinsp;+\u0026thinsp;L) secondary antibodies (Cat. No. A0208) were purchased from Beyotime (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 High-Performance Liquid Chromatography (HPLC)\u003c/h2\u003e \u003cp\u003eFIN and CUR were assayed by HPLC using a C18 column (5 \u0026micro;m, 250 mm \u0026times; 4.6 mm; Eclipse Plus; Agilent Technologies, Lexington, MA, US) coupled with a guard column (5 \u0026micro;m, 12.5 mm \u0026times; 4.6 mm) and a photodiode array detector (Infinity 1260 LC System, Agilent Technologies, Lexington, MA, US) under a gradient mode (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). The mobile phase comprised mobile phase A [0.15% TFA in UPW (v/v)] and mobile phase B [0.15% TFA in MeOH (v/v)]. A 20 \u0026micro;L sample was injected into the column at a flow rate of 1 mL/min. The UV detection wavelengths of FIN and CUR were set at 220 nm and 430 nm, respectively. FIN and CUR eluted at around 9.6 min and 5.3 min, respectively. The calibration curves showed excellent linearity for both FIN (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9997) and CUR (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9999).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Preparation of FIN Nanosuspensions\u003c/h2\u003e \u003cp\u003eThe FIN nanosuspension was produced using a four-inlet multi-inlet vortex mixer (MIVM) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cb\u003eVideo S1\u003c/b\u003e. Specifically, the drugs FIN and CUR, along with CLT, were dissolved in EtOH as an organic stream in inlet 1, while the remaining inlets (inlets 2\u0026ndash;4) were loaded with PVP aqueous solution. The flow rates of inlets 2 and 4 were regulated by a PHD ULTRA syringe pump (Harvard Apparatus, Holliston, MA, USA) at 99 ml/min, and inlets 1 and 3 were controlled by another syringe pump (Terumo Corporation, Tokyo, Japan) at 11 ml/min. The FIN nanosuspension was collected from the outlet stream of the MIVM. The flow pattern can be characterized by calculating the Reynold number (Re), as shown in Eq.\u0026nbsp;1. The resulting Re was fixed around 4,000 to ensure homogeneous mixing of the four inlet streams prior to nanoprecipitation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Re=\\sum\\:_{i=1}^{4}\\frac{{\\rho\\:}_{i}{v}_{i}d}{{\\mu\\:}_{i}}\\:=\\:\\frac{4}{\\pi\\:D}\\sum\\:_{i=1}^{4}\\frac{{\\rho\\:}_{i}{Q}_{i}}{{\\mu\\:}_{i}}\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\left(Equation\\:1\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003ei\u003c/em\u003e is the stream number, \u003cem\u003eρ\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e is the fluid density (kg/m\u003csup\u003e3\u003c/sup\u003e), \u003cem\u003eQ\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e is the stream flow rate (m\u003csup\u003e3\u003c/sup\u003e/s), \u003cem\u003e\u0026micro;\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e is the fluid viscosity (Pa\u0026acute;s), and \u003cem\u003eD\u003c/em\u003e is the internal diameter (m) of the MIVM.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Characterization of FIN Nanosuspensions\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Particle Size, Size Distribution and Zeta Potential\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ez\u003c/em\u003e-average particle size, size distribution, and polydispersity index (PDI) of FIN nanoparticles were measured by dynamic light scattering (DLS) using a Delsa Nano C particle analyzer (Beckman Coulter, Brea, CA, USA). The viscosity and refractive index of the medium were assumed to be the same as those of pure water (0.89 mPa\u0026middot;s and 1.331 at 25\u0026deg;C). The zeta potential of FIN nanoparticles was determined using the same particle analyzer mentioned above. The measured electrophoretic mobility was converted into zeta-potential using the Smoluchowski relationship [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Physical Stability\u003c/h2\u003e \u003cp\u003eThe physical stability of FIN nanosuspension was monitored by measuring the change in particle size over time [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The test was terminated when either a\u0026thinsp;\u0026gt;\u0026thinsp;20% change in particle size occurred or visible precipitation was observed in the nanosuspension.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 Encapsulation Efficiency (EE) and Drug Loading (DL) of the Nanosuspension\u003c/h2\u003e \u003cp\u003eThe determination of EE and DL was performed using an established protocol [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Briefly, 15 mL of FIN nanosuspension was transferred into an Amicon\u0026reg; Ultra 30kDa centrifugal filter device (Sigma Aldrich, St Louis, MO, USA) and centrifuged at 4000 x \u003cem\u003eg\u003c/em\u003e for 40 minutes. The filtrate, containing free FIN and CUR, was collected for HPLC analysis while the concentrated sample was freeze-dried using a Freezone 6 Liter Benchtop Freeze Dry System with Stoppering Tray Dryer (Labconco Corporation, Kansas City, MO, US). The freeze-dried product was weighed and dissolved in a mixture of UPW and MeOH with 0.15% TFA [23:77 (\u003cem\u003ev/v\u003c/em\u003e)] for HPLC analysis of the drug content in the nanoparticles. The EE and DL were then calculated according to Equations 2 and 3, respectively.\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\text{E}\\text{E}\\:\\left(\\%\\right)=\\frac{total\\:amount\\:of\\:drug-amount\\:of\\:free\\:drug}{total\\:amount\\:of\\:drug}\\times\\:100\\%\\:\\:\\:\\:\\:\\:\\:\\:Equation\\:\\left(2\\right)$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:D\\text{L}\\:\\left(\\%\\right)=\\frac{total\\:amount\\:of\\:drug\\:in\\:nanoparticles}{total\\:amount\\:of\\:nanoparticles}\\times\\:100\\%\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:Equation\\:\\left(3\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.4 Transmission Electron Microscopy (TEM)\u003c/h2\u003e \u003cp\u003eA single drop of freshly produced FIN nanosuspension was dripped on a carbon film TEM grid that had been discharged using the PELCO easiGlow\u0026trade; Glow Discharge Cleaning System (Redding, CA, US), followed by staining with 2% (\u003cem\u003ev/v\u003c/em\u003e) uranyl acetate for 1 minute. Then, the Tecnai\u0026trade; G2 20 S-TWIN Transmission Electron Microscope (FEI, Hillsboro, OR, US) was employed to image the nanoparticles on the air-dried grid at \u0026times;19,500 magnification.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Design of Experiment (DoE)-guided Optimization of FIN Nanosuspension\u003c/h2\u003e \u003cp\u003eA 2-level, 3-factor full factorial design was employed to examine the main effects and interactions of selected formulation parameters on the critical quality attributes (CQAs) of the FIN nanosuspension (\u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). The processing parameters were the initial concentration of FIN (mg/ml) (A), the mass ratio between CLT and FIN (w/w) (B), and the concentration of PVP solution [% (w/v)] (C), while the responses included particle size (nm) (Y\u003csub\u003e1\u003c/sub\u003e), PDI (Y\u003csub\u003e2\u003c/sub\u003e), physical stability (hours) (Y\u003csub\u003e3\u003c/sub\u003e), and EE of FIN (%) (Y\u003csub\u003e4\u003c/sub\u003e). The initial concentration of CUR was fixed at 2.5 mg/mL. The levels of each variable were set as +\u0026thinsp;1, 0, and \u0026minus;\u0026thinsp;1, and a total of 9 experimental runs (2\u003csup\u003e3\u003c/sup\u003e + 1 runs as the center point) were performed.\u003c/p\u003e \u003cp\u003eThe optimal processing parameters of FIN nanosuspension were identified using the desirability function approach based on the regression models constructed by Design-Expert 13 for the responses [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Each response was associated with a partial desirability function (\u003cem\u003ed\u003c/em\u003e), where a fully desired response was assigned a value of 1 and an unfavorable response was assigned a value of 0. The overall desirability value (\u003cem\u003eD\u003c/em\u003e) was determined using the geometric mean of the partial desirability functions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Preparation of FIN Nano-embedded Microparticle Dry Powder Formulations\u003c/h2\u003e \u003cp\u003eFIN nano-embedded microparticle dry powder formulations were produced using spray drying. Specifically, mannitol solution was mixed with freshly prepared optimized FIN nanosuspension, and this mixture was fed into a B\u0026uuml;chi spray dryer (Mini Spray Dryer B-290 coupled with Dehumidifier B-296, Flawil, Switzerland) with nitrogen as the drying gas [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The aspiration rate and inlet temperature were fixed at 100% (38 m\u003csup\u003e3\u003c/sup\u003e/h) and 110 ℃, while the atomization flow rate and feed rate were varied in each run according to the experimental design. The resulting product was collected in a Falcon-50 ml conical tube and transferred to a desiccator upon spray drying.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Characterization of FIN Nano-embedded Microparticle Dry Powder Formulation\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1 Aqueous Redispersibility\u003c/h2\u003e \u003cp\u003eThe redispersibility test was performed by reconstituting the spray-dried powder in UPW at room temperature. Briefly, 15 mg of dry powder was transferred into 10 ml of UPW, and the resulting suspension was stirred at 75 rpm for 10 minutes. After 3 minutes, the particle size was measured as described in Section \u003cspan refid=\"Sec7\" class=\"InternalRef\"\u003e2.4.1\u003c/span\u003e. The redispersibility index (RdI) was denoted as S\u003csub\u003ef\u003c/sub\u003e/S\u003csub\u003ei\u003c/sub\u003e, where S\u003csub\u003ei\u003c/sub\u003e and S\u003csub\u003ef\u003c/sub\u003e represent the particle size of FIN nanoparticles before and after spray drying, respectively [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. A RdI value of 1 indicates that the particle size remains unchanged during the spray drying process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e\u003cem\u003e2.7.2 Particle Size Distribution by Laser Diffraction\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eA HELOS/KR laser diffractometer (Sympatec, Germany) was used to determine the volumetric size distribution of the dry powder, as previously reported with minor modifications [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Briefly, a Unidose powder nasal spray system (Aptar Pharma, France) filled with 5.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mg dry powder was connected to the laser diffractometer (30\u003csup\u003e◦\u003c/sup\u003e angle) using an adaptor. The dry powder was then dispersed at a flow rate of 15 L/min. The spherical volume diameters at 10% (D\u003csub\u003e10\u003c/sub\u003e), 50% (D\u003csub\u003e50\u003c/sub\u003e), and 90% (D\u003csub\u003e90\u003c/sub\u003e) cumulative volumes were recorded. The span of the dry powder was expressed as (D\u003csub\u003e90\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;D\u003csub\u003e10\u003c/sub\u003e)/ D\u003csub\u003e50\u003c/sub\u003e. Each sample was measured three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.7.3 In Vitro Evaluation of Nasal Aerosol Depositions\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section4\"\u003e \u003ch2\u003e2.7.3.1 3D-printed Nasal Cast Model\u003c/h2\u003e \u003cp\u003eAs depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, a Next Generation Impactor (NGI) (Copley, Nottingham, UK) coupled with a customized 3D-printed nasal cast model was employed to assess the \u003cem\u003ein vitro\u003c/em\u003e aerosol performance of the optimized FIN dry powder formulation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The 3D-printed nasal cast can be dismantled into two parts: the olfactory region and the respiratory region. A thin layer of silicon grease (Slipicone; LPS Laboratories, Tucker, GA, USA) was sprayed on all stages of the NGI to minimize particle bouncing. An unidose powder nasal spray device (Aptar Pharma, France) was used to disperse 15.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 mg of optimized FIN dry powder into the nasal cavity under inspiratory flow rates of 0, 7.5, and 15 L/min. The insertion depth and the insertion angle of the nasal device into the nostril were 5 mm and 60\u003csup\u003e◦\u003c/sup\u003e from the horizontal plane, respectively. After dispersion, the powder in the 3D-printed nasal cast model and NGI stages was rinsed using the HPLC mobile phase (UPW and MeOH with 0.15% TFA [23:77 (v/v)]). The resulting solution was filtered through a 0.45-\u0026micro;m membrane for FIN assay. The deposition experiment was repeated thrice.\u003c/p\u003e \u003cp\u003eThe recovered dose was defined as the total mass of FIN recovered in the 3D-printed nasal cast model and the NGI stages, and all fractions were calculated with respect to the recovered dose. The nasal device fraction was calculated with the powder mass remaining in the nasal spray device after dispersion, while the throat fraction was calculated using the powder mass deposited in the throat and adaptor regions connecting the NGI and nasal cast. The NGI stages fraction was calculated based on the powder mass deposited in Stages 1\u0026ndash;7 and the MOC of the NGI.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section4\"\u003e \u003ch2\u003e2.7.3.2 Alberta Idealised Nasal Inlet (AINI) Model\u003c/h2\u003e \u003cp\u003eAn Alberta Idealised Nasal Inlet (AINI) (Copley, Nottingham, UK) coupled to the NGI was also utilized to examine \u003cem\u003ein vitro\u003c/em\u003e nasal aerosol deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This model was created based on a set of realistic nasal anatomies and composed of four detachable components, i.e., olfactory region, vestibule, turbinates, and nasopharynx [\u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The procedures for coating the NGI stages, filling the nasal spray device, inserting the nasal spray device, FIN assay, and calculations of the recovered dose and nasal device fraction were the same as those described in Section \u003cspan refid=\"Sec17\" class=\"InternalRef\"\u003e2.7.3.1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e2.7.4 Scanning Electron Microscopy\u003c/h2\u003e \u003cp\u003eA Hitachi S-4800 FEG field emission scanning electron microscope (Hitachi, Tokyo, Japan) was utilized to characterize the particle morphology of the spray-dried powder at 5.0 kV. The powder sample was gently dispersed on carbon tape mounted on a SEM stub and subsequently coated by an ~\u0026thinsp;11 nm gold-palladium alloy using a sputter coater for 90 seconds to prevent charging interferences during the imaging.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e2.7.5 Powder X-Ray Diffractometry (PXRD)\u003c/h2\u003e \u003cp\u003eA Rigaku SmartLab 9 kW diffractometer with a copper rotating anode (K alpha1 1.54059 \u0026Aring;, K alpha2 1.54441 \u0026Aring;) rated at 160 mA/ 45 kV was used to collect the PXRD patterns of the samples. A K beta nickel filter was used to filter the diffraction signals. Each sample was scanned within a 2θ range of 3\u0026deg; to 40\u0026deg;, with a step width of 0.02\u0026deg; and a scanning speed of 5.0\u0026deg; per minute.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e2.7.6 Fourier-Transform Infrared Spectroscopy (FTIR)\u003c/h2\u003e \u003cp\u003eA Spectrum Two FTIR spectrometer (Perkin Elmer, Waltham, MA, USA) was used to generate FT-IR spectra in KBr diffuse reflectance mode. The scan was performed in the range of 4,000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 1,000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at intervals of 0.5 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. A total of 32 scans were performed at a resolution of 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e2.7.7 Differential Scanning Calorimetry (DSC)\u003c/h2\u003e \u003cp\u003eThe thermal characteristics of the samples were measured using a DSC 250 differential scanning calorimeter (TA Instruments, New Castle, DE, USA). Prior to the measurement, pure indium was used for calibration. Each sample (3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mg) was encased in a Tzero hermetic pan and heated from 50\u0026deg;C to 250\u0026deg;C at a ramp rate of 10\u0026deg;C/min under an N\u003csub\u003e2\u003c/sub\u003e flow rate of 20 mL/min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e2.7.8 Thermogravimetric Analysis (TGA)\u003c/h2\u003e \u003cp\u003eThe residual solvent and moisture content (\u003cem\u003eM\u003c/em\u003e) of the spray-dried powder was determined using a TGA550 thermogravimetric analyzer (TA Instruments, Newcastle, DE, USA) according to Eq.\u0026nbsp;(5). Approximately 3 mg of sample was loaded in a platinum pan and heated from 25\u0026deg;C to 200\u0026deg;C at a scanning rate of 10\u0026deg;C/min under a N\u003csub\u003e2\u003c/sub\u003e flow rate of 20 mL/min.\u003cdiv id=\"Equd\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e\n$$\\:M\\:\\left(\\%\\right)=\\frac{{m}_{0}-{m}_{1}}{{m}_{0}}\\times\\:100\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:Equation\\:\\left(4\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere m\u003csub\u003e0\u003c/sub\u003e and m\u003csub\u003e1\u003c/sub\u003e represent the weight of the measured sample before and after the experiment, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e2.7.9 Encapsulation Efficiency (EE) and Drug Content of Powders\u003c/h2\u003e \u003cp\u003eTo determine the encapsulation efficiency of reconstituted nanosuspension, 20 mg of spray-dried dry powder was dispersed into 10 ml of UPW under a stirring rate of 75 rpm for 10 minutes. The reconstituted nanosuspension was subsequently transferred into the filter device (Amicon\u0026reg; Ultra-15, Sigma Aldrich, St Louis, MO, USA) and centrifuged at 4000 x g for 40 min. The analytical procedure was the same as mentioned in section \u003cspan refid=\"Sec9\" class=\"InternalRef\"\u003e2.4.3\u003c/span\u003e. Regarding the drug content of FIN and CUR in the spray-dried dry powder, 10 mg of sample was accurately weighed and transferred into the tube and dissolved in a 2 ml mixture of UPW and MEOH with 0.15% TFA [23:77 (\u003cem\u003ev/v\u003c/em\u003e)] for HPLC assay of FIN and CUR in the sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e2.7.10 Stability Studies\u003c/h2\u003e \u003cp\u003eThe samples were stored in screw-capped glass tubes at 4\u0026deg;C, room temperature, and 40\u0026deg;C under 30% relative humidity for 2 months. The chemical stability of the sample was monitored by conducting HPLC assays of FIN and CUR, while the physical stability of the sample was checked by the DSC, TGA, and PXRD analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e2.7.11 In Vitro Drug Release\u003c/h2\u003e \u003cp\u003eThe drug release profiles of the samples were obtained using a reported protocol with modification [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. 50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mg of powder samples or its physically mixed counterpart was dispersed into 20 ml of simulated nasal fluid, and the resulting solution was stirred at 75 rpm for 3 hours at 34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u0026deg;C [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. 0.5 ml of solution was withdrawn at designated time points (15, 30, 60, 120, and 180 minutes) and transferred into an Amicon\u0026reg; Ultra-0.5 centrifugal filter unit. Medium replacement was carried out upon each sample collection. The FIN content in the filtrate was assayed by HPLC. The experimental procedure was repeated with the change of the medium to a mixture of simulated nasal fluid and ethanol [80:20 (v/v)].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e2.8 DoE-guided Optimization of FIN Nano-embedded Dry Powder Formulation\u003c/h2\u003e \u003cp\u003eThe experimental design, optimization, and model validation of the fabrication of FIN dry powder formulation were the same as the FIN nanosuspension production described in Section \u003cspan refid=\"Sec11\" class=\"InternalRef\"\u003e2.5\u003c/span\u003e. Herein the formulation and processing parameters in the DoE (\u003cb\u003eTable S.3.\u003c/b\u003e) included the ratio between mannitol and nanoparticle (w/w) (A), the atomization gas flow rate (L/h) (B), and the feed rate of solution during the spray drying process (ml/min) (C), while the responses included redispersibility of the dry powder (Z\u003csub\u003e1\u003c/sub\u003e), PDI of the reconstituted nanosuspension (Z\u003csub\u003e2\u003c/sub\u003e), and volumetric particle size of dry powder (\u0026micro;m) (Z\u003csub\u003e3\u003c/sub\u003e). Again, the optimal processing parameters of FIN co-loaded dry powder were identified by the desirability function approach based on the regression models constructed by Design-Expert 13 for the responses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e2.9 \u003cem\u003eIn Vitro\u003c/em\u003e Cellular Study\u003c/h2\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003e2.9.1 Cell Culture\u003c/h2\u003e \u003cp\u003eSH-SY5Y cells were cultured in DMEM culture medium, and PC 12 cells were cultured in F-12K nutrient mixture medium containing L-glutamine. RPMI 2650 cells were cultured in MEM, while Calu-3 cells were cultured in DMEM/F12 culture medium. These cell culture mediums were supplemented with 10% (v/v) FBS and 1% (v/v) antibiotic-antimycotic. The cells were cultured in a humidified incubator at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e and were passaged in different ratios when the cell density reached 80% confluence. 0.25% trypsin-EDTA was used to detach the cells from the T75 flasks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section3\"\u003e \u003ch2\u003e\u003cem\u003e2.9.2 In Vitro Cellular Viability Assay\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eCells were seeded in 96-well plates at a density of 2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well (for RPMI 2650 and PC-12), 0.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well (for SH-SY5Y), or 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well (for Calu-3) and cultured in a humidified incubator at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e overnight. On day 2, the cells were treated with reconstituted optimized FIN dry powder, raw FIN, and a physical mixture of FIN powder formulation components at FIN concentrations ranging from 2.5 to 1000 nM for 24 hours. The reconstituted optimized FIN dry powder was prepared by redispersing optimized FIN dry powder in the cell culture medium. Raw FIN and their physical mixture were dissolved in DMSO and further diluted by the cell culture medium to a desired concentration. Considering the cytotoxicity of DMSO, cells were also treated with raw DMSO, which was the same as the DMSO concentration in the treatment groups as the control group. After 24 hours, the cells were incubated with 100 \u0026micro;L of cell culture medium with 10% CCK-8 solution for 3 hours. The spectrophotometric absorbance of the sample was measured at 450 nm using a microplate spectrophotometer (MultiSkan Go, Thermo Fisher Scientific, Waltham, MA, USA). The cell viability was calculated as the percentage of the absorbance from the treatment groups divided by the absorbance from the control group. All measurements were performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e2.10 \u003cem\u003eIn Vivo\u003c/em\u003e Animal Study\u003c/h2\u003e \u003cdiv id=\"Sec32\" class=\"Section3\"\u003e \u003ch2\u003e2.10.1 Animals and Ethics Statements\u003c/h2\u003e \u003cp\u003e All animal studies followed the ethical policies and guidelines recommended by ARRIVE (Animal Research: Reporting of In Vivo Experiments) and National Research Council's Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of the Animal Experimentation of Jinan University (Approval No.: 20230901-0012). Male C57BL/6 mice (6\u0026ndash;8 weeks old) were obtained from Zhuhai Baishitong Biotechnology Co., Ltd and were used for the ischemic stroke models. All mice were kept in a daily cycle of 12 hours of light and 12 hours of darkness with humidity and temperature controls. Water and food were freely available to the mice at all times in their cages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003e2.10.2 Development of the Ischemic Stroke Model\u003c/h2\u003e \u003cp\u003eElectrocoagulation was used to create the middle cerebral artery occlusion (MCAO) model based on a reported protocol on day 1 [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Mice were anaesthetized using 1% (w/v) pentobarbital sodium at a dose of 50 mg/kg by intraperitoneal injection. Subsequently, a skin incision was made between the ear and right eye to expose the skull and temporalis muscle. The middle cerebral artery was exposed by penetrating the skull with a microdrill. Finally, a small vessel electrocoagulator was utilized to cauterize the distal middle cerebral artery. Following the restoration of the temporalis, a surgical suture was used to close the wound in the head skin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003e2.10.3 Animal Grouping\u003c/h2\u003e \u003cp\u003eA total of 32 mice were randomly divided into 4 groups (n\u0026thinsp;=\u0026thinsp;8 per group): (1) sham-operated group (Sham group); (2) MCAO group receiving 0.9% saline solution intranasally (IN) (MCAO group); (3) MCAO group treated with the optimized FIN dry powder (1 mg/kg of FIN) reconstituted in 0.9% saline solution via intravenous (IV) injection (MCAO\u0026thinsp;+\u0026thinsp;IV FIN group); and (4) MCAO group treated with the optimized FIN dry powder (1 mg/kg of FIN) reconstituted in 0.9% saline solution (~\u0026thinsp;15 \u0026micro;L) intranasally (IN) (MCAO\u0026thinsp;+\u0026thinsp;IN FIN group). The initial dose was administered 30 minutes following the ischemic stroke surgery, and the second dose was given 24 hours post-surgery. On the third day, the mice were sacrificed, and brain tissues were collected for western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec35\" class=\"Section3\"\u003e \u003ch2\u003e2.10.4 Behavioural Assessments and Neurological Function Evaluation\u003c/h2\u003e \u003cp\u003eThe neurological recovery of mice was assessed using the adhesive removal test balance beam test, and rotarod test on days 2 and day 3 [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Baseline behavioural assessment was conducted one day prior to the stroke surgery. In the adhesive removal test, mice were placed in a container, and the time for the mice to remove the adhesive tapes from the bilateral paws was recorded. For the balance beam test, the time taken for the mice to walk across a beam (1.20 m in length, 1.0 cm in width, and 0.7 m in height) was recorded after training. In the rotarod test, mice were trained at constant speed three times before the surgery and the retention time for mice to remain on the rotarod with accelerated speed (starting at 4 rpm and increasing by 46 rpm per 60 seconds until the 50 rpm) was assessed. Furthermore, the Longa score was used to assess the neurological deficits of mice on day 3, especially the motor function of mouse [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The weight of mice was monitored daily.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec36\" class=\"Section3\"\u003e \u003ch2\u003e2.10.5 Histological Assessment\u003c/h2\u003e \u003cp\u003eThe histological assessments were conducted on day 2 after the behavioural assessments and neurological function evaluation as previously described [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Briefly, the mice were anesthetized (1% pentobarbital sodium, 50 mg/kg) and were perfused with 0.9% sodium chloride transcardially. After fixing with 4% paraformaldehyde, the brain was removed rapidly and dipped into the wax, followed by embedding and sectioning in 4 \u0026micro;m thickness. The brain slice was observed at the posterior and anterior levels based on Paxinos and Franklin's mouse brain in stereotaxial coordinates. The brain slices were defatted by xylene and dehydrated by alcohol. For Nissl staining, the brain slices were immersed in 0.1% cresol violet for 30 minutes at room temperature. The brain slices were dehydrated after rinsing by UPW, followed by transparentizing and sealing with neutral gum. The TissueFAXS PLUS system from TissueGnostics was employed to scan slides automatically.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec37\" class=\"Section3\"\u003e \u003ch2\u003e2.10.6 Magnetic Resonance Imaging (MRI)\u003c/h2\u003e \u003cp\u003eThe MRI was conducted on day 2 using a reported protocol with modification [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The 9.4 T small animal MRI scanner (Bruker PharmaScan) was used to obtain MRI images of the mice 24 hours after the stroke surgery. The parameters of the T2-weighted imaging (T2WI) imaging were set as a field of view (FOV)\u0026thinsp;=\u0026thinsp;20 \u0026times; 20 mm, 17 axial slices with a slice thickness of 1 mm, a matrix of 256 \u0026times; 256 and 2D fast-spin echo sequence (3500/33 ms of repetition time/echo time, 2 average). After anesthetization, the respiratory rate and the temperature of mice were monitored during the imaging process. The machine was placed over the brains of mice, followed by the scanning by T2WI imaging and quantitively analysis using a 3D slicer software. Based on the T2WI images, the 3D slicer was used to reconstruct the 3D images. The infarct and non-infarct zones were separated using threshold modification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e \u003ch2\u003e2.10.7 Western Blot Analysis\u003c/h2\u003e \u003cp\u003eThe western blot analysis was conducted on day 3 according to a reported protocol with modifications [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The RIPA buffer was used for lysing and extracting proteins from the mouse brain. Protein samples (5\u0026ndash;30 \u0026micro;g/lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene difluoride membrane (Bio-Rad). The membrane was blocked using TBST buffer containing 5% milk for one hour, followed by overnight incubation with primary antibodies against CC3, BCL-2, BAX, and β-Tubulin (as internal control) proteins at 4℃ in 5% milk-TBST. The blot was then washed and incubated with secondary antibodies in TBST buffer containing 5% milk for one hour at room temperature. The ChemiDoc Touch imaging system (Bio-Rad) was used to detect the protein bands, while Image Lab software and Tanon 2500 Gel Imaging System were employed for data analysis. Protein bands were analyzed and quantified using Quantity One (Bio-Rad) or ImageJ with normalization provided by the respective loading controls in the same samples.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec39\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistical Analysis\u003c/h2\u003e \u003cp\u003eData were shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (S.D.). The optimal processing parameters of FIN nanosuspension and dry powder were obtained by Design-Expert (Version 21.3.1; Stat-Ease, Inc., Minneapolis, USA) using least-square regression and nested ANOVA of the responses. Trios (Version 5.1.1; TA Instrument, New Castle, DE, USA) was used for the analysis of DSC data. A \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec41\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Optimization of FIN Nanosuspensions\u003c/h2\u003e \u003cp\u003eThe selection of formulation parameters and the corresponding design space (\u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e) in the factorial design for fabricating FIN nanosuspensions were guided by preliminary data and technical considerations for nose-to-brain drug delivery. Without the aid of stabilizers, the resulting nanosuspensions exhibited visible precipitation within 5 minutes right after production, while the incorporation of PVP and CLT into the formulations significantly enhanced the physical stability of nanosuspensions (\u003cb\u003eTable S4\u003c/b\u003e). Regarding the response specifications, as the diameter of olfactory axon in humans falls within a typical range of 100\u0026ndash;700 nm, and previous research has demonstrated that nanoparticles with a size of less than 200 nm can be transported effectively to the brain via transcellular neuronal absorption, the target size for FIN nanoparticles was set in the range of 0-200 nm [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In addition, considering that the nanosuspensions would be spray-dried into dry powders, they should remain colloidally stable before the drying process. Therefore, the physical stability was set as over 24 hours, allowing sufficient transit time in real manufacturing practice. To unleash the therapeutic potential of the formulations, targets for the EE of FIN and PDI of the nanoparticles were set in line with the literature standards at 90% and \u0026lt;\u0026thinsp;0.3, respectively [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough single-factor experiments are frequently used for optimizing drug formulations and manufacturing processes, it is relatively time-consuming and incapable of identifying potential interaction effects of factors on responses [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Consequently, a full factorial DoE was employed in this study. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the physicochemical properties of FIN nanosuspensions obtained from the DoE. The particle size of the nanosuspensions ranged from 107.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4 nm to 187.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 nm, which were all within the target range. The PDI of these formulations, except for NF2, satisfied the pre-set requirement of \u0026lt;\u0026thinsp;0.3. Notably, there was a discrepancy in the stability among these formulations. For example, NF2 and NF5 showed precipitation within 30 minutes, whereas NF3 remained stable for at least 72 hours. The EE of FIN among these formulations varied from 60.51\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84% to 95.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04%, while the EE of CUR consistently surpassed 99.99%. The coded regression equations for each response model of the FIN nanosuspensions are shown in \u003cb\u003eTable S5\u003c/b\u003e. These models were tested by two-way nested ANOVA and displayed statistical significance (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) for all responses, suggesting that the factors and their interactions were significantly correlated with the responses. They were also considered reliable with predicted \u003cem\u003eR\u003c/em\u003e\u0026sup2; values close to or exceeding 0.85, which agreed with the adjusted \u003cem\u003eR\u003c/em\u003e\u0026sup2; values. All regression models were therefore used to probe the relative effects of factors and their interactions on the responses.\u003c/p\u003e \u003cp\u003eThe Pareto charts in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e show the standardized effects of factors and their interactions on the responses. All studied factors and their interactions exerted a significant effect on the particle size of FIN nanosuspensions (Y\u003csub\u003e1\u003c/sub\u003e). Specifically, increasing the initial FIN concentration (A), CLT: FIN ratio (B), the concentration of PVP solution (C), and their interaction terms AC and BC tended to produce larger particles. The observations in the main effects could be partly explained by the nucleation and growth theories. Upon bulk mixing of the aqueous and organic streams in the MIVM, an upsurge in the supersaturation of hydrophobic solutes (FIN, CUR, and CLT) would concurrently increase their nucleation and particle growth rates, resulting in a contrasting effect on the particle size. Quenching further particle growth requires adequate deposition of the water-soluble stabilizer (PVP) on nanoparticles (i.e., nuclei with dimensions beyond the critical size) via molecular diffusion, which is a time-dependent process [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. This explains why a sole rise in factor A or B led to an increase in particle size, as the resulting nanoparticles were not instantly stabilized by PVP, and their effects on particle growth dominated that of nucleation. It is worth noting that factor C could influence particle size in both negative and positive ways. While PVP could inhibit nanoparticle growth and aggregation by surface capping, it also acted as a solubilizing agent to alter the supersaturation levels of FIN and CLT in the systems and, consequently, their nucleation kinetics. The latter effect was more apparent in the current design space. The dual roles of PVP also caused factor C to have a lower \u003cem\u003et\u003c/em\u003e-value in two-way nested ANOVA and coefficient in the regression equation compared to the other main factors. Surprisingly, the interaction terms AB and ABC, albeit marginally statistically significant, were negatively correlated with particle size. We speculate that when both factors A and B increased, the precipitated hydrophobic CLT was able to shorten the diffusion path and time of PVP from the bulk solution to the nuclei, thereby yielding smaller nanoparticles. However, the molecular interplays in nanoparticle formation and stabilization for a multi-component system are complex, and further research (e.g. using molecular dynamics simulation) is warranted to delineate their exact mechanisms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePertaining to the PDI (Y\u003csub\u003e2\u003c/sub\u003e), physical stability (Y\u003csub\u003e3\u003c/sub\u003e), and EE of FIN (Y\u003csub\u003e4\u003c/sub\u003e) responses, the factor-response relationships elucidated from the models generally aligned with the findings derived from the response model of particle size (Y\u003csub\u003e1\u003c/sub\u003e) and our expectations. As mentioned earlier, augmenting factor A or B would elevate the supersaturation level of the hydrophobic solutes, thereby facilitating the entrapment of more FIN molecules within the nanoparticle core during the FNP process. The resulting larger nanoparticles also possessed lower surface energy, which served to minimize particle aggregation and produce a more monodisperse nanosuspension. It is not surprising that factor C and its related 2-factor interactions (i.e., AC and BC) displayed erratic effects on the PDI, EE, and stability since PVP serves multiple functions as a crystallization inhibitor, solubilizer, and stabilizer in the nanosuspension formulation as aforementioned. Briefly, factor C had a positive, negative, and insignificant effect on PDI, EE, and stability, respectively, but its interaction factors AC and BC showed divergent trends (AC: negative for PDI, positive for EE and stability; BC: insignificant for PDI and stability, positive for EE). It should be noted that these regression models are of good predictive ability within our design space but their applicability to other systems may be limited, particularly if the design spaces of those systems differ significantly from the present study.\u003c/p\u003e \u003cp\u003eThe regression models were simplified by eliminating non-statistically significant terms (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and the desirability function was applied to optimize the FIN nanosuspension, and the highest desirability value (0.951) was obtained under the condition of A\u0026thinsp;=\u0026thinsp;5 (mg/mL), B\u0026thinsp;=\u0026thinsp;1.5 (\u003cem\u003ew/w\u003c/em\u003e) and C\u0026thinsp;=\u0026thinsp;0.25 (% \u003cem\u003ew/v\u003c/em\u003e). Additional experimental runs (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3) using such parameters were performed for model validation. The resulting nanosuspensions had a particle size of 134.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 nm (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea\u003c/b\u003e), PDI of 0.179\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021, physical stability of 72\u0026thinsp;\u0026plusmn;\u0026thinsp;0 hours, and EE of FIN of 90.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08%, which were all in line with the predictions (particle size of 133.83 nm, PDI of 0.161, physical stability of 72 hours, and EE of FIN of 91.07%). The DLs of FIN (8.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25%) and CUR (4.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15%) were well aligned with the theoretical values, i.e., 8% for FIN and 4% for CUR. The zeta potential of the optimized nanosuspension was \u0026minus;\u0026thinsp;0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 mV, suggesting its near-neutral state. As shown in \u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb\u003c/b\u003e, FIN nanoparticles displayed roughly spherical morphology with similar size to that of DLS. As a result, the optimized FIN nanosuspension was used for the subsequent development of nano-embedded microparticle dry powders.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysicochemical properties of FIN nanosuspensions prepared based on the 3-factor 2-level factorial design (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNanosuspension\u003c/p\u003e \u003cp\u003eFormulation (NF)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eFactors with levels\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c8\" namest=\"c5\"\u003e \u003cp\u003eResponses and results\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eParticle Size (nm) (Y\u003csub\u003e1\u003c/sub\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePDI (Y\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePhysical Stability (hours) (Y\u003csub\u003e3\u003c/sub\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eEE of FIN (%) (Y\u003csub\u003e4\u003c/sub\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e136.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.238\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e88.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNF2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e107.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.381\u0026thinsp;\u0026plusmn;\u0026thinsp;0.072\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e60.51\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNF3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e134.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.179\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e72.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e90.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNF4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e142.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.251\u0026thinsp;\u0026plusmn;\u0026thinsp;0.044\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e40.0\u0026thinsp;\u0026plusmn;\u0026thinsp;13.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e68.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNF5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e155.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.171\u0026thinsp;\u0026plusmn;\u0026thinsp;0.019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e95.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNF6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e166.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.208\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e81.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNF7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e151.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.181\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e6.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e95.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNF8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e187.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.186\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e24.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e85.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNF9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.625\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e154.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.205\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e6.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e84.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eA: Initial FIN concentration (mg/ml); B: CLT: FIN (w/w); C: Concentration of PVP solution (% w/v)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCoded regression equations of response models for optimization of FIN nanosuspension with ANOVA and multiple reliability test (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e) results.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResponse\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRegression equation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAdjusted R\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParticle Size (nm) (Y\u003csub\u003e1\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=\u0026thinsp;147.68\u0026thinsp;+\u0026thinsp;17.48A\u0026thinsp;+\u0026thinsp;6.28B\u0026thinsp;+\u0026thinsp;3.26C\u0026thinsp;+\u0026thinsp;8.4AC\u0026thinsp;+\u0026thinsp;7.78BC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e128.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.970\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.962\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePDI (Y\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=\u0026thinsp;0.2244-0.0377A-0.0252B\u0026thinsp;+\u0026thinsp;0.0321C\u0026thinsp;+\u0026thinsp;0.0222AB-0.0218AC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.812\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.765\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhysical Stability (hours) (Y\u003csub\u003e3\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=\u0026thinsp;18.00-10.17A\u0026thinsp;+\u0026thinsp;17.50B-1.67C-10.33AB\u0026thinsp;+\u0026thinsp;6.50AC-1.83BC\u0026thinsp;+\u0026thinsp;6.00ABC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e95.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.974\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.964\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEE of FIN (%) (Y\u003csub\u003e4\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=\u0026thinsp;83.22\u0026thinsp;+\u0026thinsp;6.10A\u0026thinsp;+\u0026thinsp;1.75B-9.36C-0.6796AB\u0026thinsp;+\u0026thinsp;3.29AC\u0026thinsp;+\u0026thinsp;1.13BC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e500.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.994\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.992\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eNote: Factors BC and C in the equation for Y\u003csub\u003e3\u003c/sub\u003e were retained to maintain the hierarchy despite being statistically non-significant.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec42\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Optimization of FIN Dry Powder Formulations\u003c/h2\u003e \u003cp\u003eAs with the optimization of FIN nanosuspension, a 2-level, 3-factor full factorial DoE was used to study the influence and interactions of factors [A: mannitol-nanoparticle ratio (w/w); B: atomization flow rate (L/h); and C: feed rate (ml/min)] on the selected properties of the resulting spray-dried nano-embedded powders [Z\u003csub\u003e1\u003c/sub\u003e: redispersibility (RdI); Z\u003csub\u003e2\u003c/sub\u003e: PDI of the reconstituted nanosuspension; and Z\u003csub\u003e3\u003c/sub\u003e: volumetric size of the spray-dried powder] (\u003cb\u003eTable S3\u003c/b\u003e). Mannitol was employed as a carrier in spray drying studies due to its promising safety for pulmonary delivery [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. To preserve the merits of nanoparticles for intranasal delivery, the spray-dried powder should be redispersible to its nanosuspension counterpart, ideally with the same particle size distribution as that freshly prepared from FNP, once in contact with the nasal fluid. Hence, the RdI was set in the range of 0.8\u0026ndash;1.2, and the PDI of reconstituted nanosuspension was set as \u0026lt;\u0026thinsp;0.3. In addition, the particle size of dry powder is a pivotal factor in controlling their nasal deposition profile. Particles less than 5 \u0026micro;m tend to enter the respiratory tract, while particles larger than 20 \u0026micro;m remain in the anterior part of the nose [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Only powder with a particle size around 10 \u0026micro;m can effectively deposit in the olfactory region [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The target median particle size of the spray-dry powder was therefore set as 10 \u0026micro;m. The levels of each factor were determined based on previous studies [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] and our preliminary data.\u003c/p\u003e \u003cp\u003eThe physicochemical properties and volumetric particle size distribution of different FIN dry powder formulations obtained from the DoE were presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cb\u003eTable S6\u003c/b\u003e, respectively. Their RdI varied from 1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 to 1.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08, and the PDI of the reconstituted nanosuspensions were close to 0.3. Among different formulations, PF1, PF6, PF7, and PF9 satisfied the pre-set RdI and PDI requirements. The volumetric particle sizes of these dry powder formulations ranged from 4.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57 \u0026micro;m to 10.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47 \u0026micro;m with span\u0026thinsp;\u003cem\u003e\u0026lt;\u003c/em\u003e\u0026thinsp;2 (except for PF3). The volumetric median particle size of these formulations, except for PF3 and PF4, was close to the pre-set 10 \u0026micro;m. The EEs of FIN and CUR for all dry powder formulations were all \u0026gt;\u0026thinsp;90%. The coded regression equations for each response model of FIN dry powder formulations are listed in \u003cb\u003eTable S7\u003c/b\u003e. The regression equations for redispersibility and particle size had \u003cem\u003ep\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, demonstrating a significant relationship between factors and their interactions with the responses. These models are considered reliable as their \u003cem\u003eR\u003c/em\u003e\u0026sup2; values were \u0026gt;\u0026thinsp;0.90 and agreed with the adjusted R\u0026sup2;. However, the \u003cem\u003ep-\u003c/em\u003evalue and \u003cem\u003eR\u003c/em\u003e\u0026sup2; values for the PDI regression are 0.715 and 0.200, respectively, indicating that there was no significant factor-response correlation. Hence, the PDI was not included in the numerical optimization process.\u003c/p\u003e \u003cp\u003eThe Pareto charts of standardized effects of factors and their interactions on the responses are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. All factors and their interactions, except for AC and ABC, showed a positive influence on the RdI of the dry powder. When factor A (mannitol-nanoparticle ratio) increased, the total solute concentration and drying time of nanosuspension droplets would also increase, leading to adverse outcomes on the nanoparticle stability and RdI. The most significant factor affecting RdI was factor B (atomization flow rate) in this study. This was not surprising and could be ascribed to greater shear stresses exerted on the nanoparticle surface during feed atomization. Moreover, a rise in factor C (feed rate) would generate larger droplets, resulting in a longer drying time and stronger thermal stress on the nanoparticles, hence the corresponding increase in RdI. The interaction terms AB and BC also shared the same trend and effect on RdI with their individual factors. Interestingly, all factors and their interactions did not have a significant effect on the PDI of the reconstituted nanosuspension. This implied that these factors had a minimal impact on the uniformity of nanoparticles embedded in the resulting spray-dried powders. Regarding the particle size of the powder, both factors A and B and their interactions AB and BC displayed a negative effect. An increase in factor B would yield smaller droplets and, thus, smaller particles. However, the trend for factor A deviated from literature reports, where particle size increases as solute concentration in feed solution increases [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. We speculate that the denser dry powders (PF3 and PF4) produced by high solute concentrations with low atomization flow rates were fractured during the dispersion from the nasal device, resulting in a smaller volumetric particle size.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe regression models for redispersibility and particle size were simplified by removing non-significant terms (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), and the desirability function was applied to optimize the FIN nano-embedded powder formulation. A moderate level of drug loading can reduce medication dosage. Since an increase in factor A (mannitol-nanoparticle ratio) is correlated with increasing RdI, and it is preferable to minimize the excipients used, the level of A is set as the lowest in the design space. The highest desirability value was achieved when A\u0026thinsp;=\u0026thinsp;4:1 (\u003cem\u003ew/w\u003c/em\u003e), B\u0026thinsp;=\u0026thinsp;407 (L/h), and C\u0026thinsp;=\u0026thinsp;4.2 (mL/min), and the corresponding predicted RdI and volumetric median particle size of the dry powder were 1.1 and 10 \u0026micro;m, respectively. Model validation was performed for fabricating the FIN dry powder (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3) using the optimized parameters. The obtained dry powder had a RdI of 1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 and a median particle size of 11.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 \u0026micro;m, congruent with the predicted properties. The PDI (0.272\u0026thinsp;\u0026plusmn;\u0026thinsp;0.019) of the nanosuspension reconstituted from the dry powders also fulfilled the target requirement. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, the optimized dry powder could be reconstituted back to nanosuspension without significant changes in particle size distribution compared to the freshly prepared nanosuspension. The volumetric size of the dry powder was 11.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 \u0026micro;m, which was expected to be effectively deposited in the olfactory region of the nasal cavity. The drug contents of FIN (1.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09%) and CUR (0.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11%) of the dry powder were consistent with the theoretical values (i.e., 1.6% for FIN and 0.8% for CUR).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysicochemical properties of FIN dry powders prepared based on the 3-factor 2-level factorial design (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePowder\u003c/p\u003e \u003cp\u003eFormulation\u003c/p\u003e \u003cp\u003e(PF)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eFactors with levels\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eResponses and results\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRdI(Z\u003csub\u003e1\u003c/sub\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePDI (Z\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eParticle Size D\u003csub\u003e50\u003c/sub\u003e (\u0026micro;m) (Z\u003csub\u003e3\u003c/sub\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e601\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.299\u0026thinsp;\u0026plusmn;\u0026thinsp;0.044\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e8.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePF2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e601\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.347\u0026thinsp;\u0026plusmn;\u0026thinsp;0.092\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e9.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePF3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e357\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.338\u0026thinsp;\u0026plusmn;\u0026thinsp;0.061\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e5.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePF4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e357\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.310\u0026thinsp;\u0026plusmn;\u0026thinsp;0.020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e4.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePF5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e357\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.303\u0026thinsp;\u0026plusmn;\u0026thinsp;0.038\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e8.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePF6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e357\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.293\u0026thinsp;\u0026plusmn;\u0026thinsp;0.038\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e10.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePF7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e601\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.285\u0026thinsp;\u0026plusmn;\u0026thinsp;0.030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e8.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePF8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e601\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.305\u0026thinsp;\u0026plusmn;\u0026thinsp;0.029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e7.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePF9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e473\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.298\u0026thinsp;\u0026plusmn;\u0026thinsp;0.020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e8.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eA: Mannitol: nano (w/w); B: Atomization gas flow rate (L/h); C: Feed rate (ml/min)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCoded regression equations of response models for optimization of FIN dry powders with ANOVA and multiple reliability test (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e) results.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResponse\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRegression equation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAdjusted R\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRedispersibility (Z\u003csub\u003e1\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=\u0026thinsp;1.26\u0026thinsp;+\u0026thinsp;0.1150A\u0026thinsp;+\u0026thinsp;0.1850B\u0026thinsp;+\u0026thinsp;0.0475C\u0026thinsp;+\u0026thinsp;0.1558AB\u0026thinsp;+\u0026thinsp;0.0717BC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e170.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.977\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.971\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVolumetric Particle Size of dry powder (\u0026micro;m) (Z\u003csub\u003e3\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e=\u0026thinsp;7.99-0.9046A-1.61B\u0026thinsp;+\u0026thinsp;0.2029C-0.6063AB-0.5738BC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e62.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.940\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.925\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eNote: Factor C in the equation for Z\u003csub\u003e3\u003c/sub\u003e was retained to maintain the hierarchy despite being statistically non-significant.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec43\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Characterization of the FIN Nano-Embedded Dry Powder Formulation\u003c/h2\u003e \u003cp\u003ePresented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec are the SEM images of the optimized FIN dry powders. The particles exhibited a spherical morphology with embedded nanoparticles (as indicated by the black frame), contributing to their good flowability for ease of powder loading in nasal spraying devices. Considering that a higher aspect ratio of particles could augment powder deposition in the alveolar region, the spherical morphology minimizes the fraction of particles inadvertently deposited in deep lung areas during intranasal application [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. As seen in the DSC and PXRD profiles (\u003cb\u003eFigs. S2a and S2b\u003c/b\u003e), except PVP, all other raw materials, their physical mixture counterpart, and the optimized FIN dry powder were crystalline in nature. The TGA analysis revealed that approximately 3.01% of residual moisture was present in the optimized dry powders, and its moisture content gave a temperature-dependent reduction to 0.91\u0026ndash;1.40% upon 1 month of storage (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ec\u003c/b\u003e). This was not surprising as unbound water in the powder evaporated with a moisture balance. The optimized FIN dry powder possessed satisfactory stability as no significant phase transformation and reduction in drug assays were detected under various storage conditions. Presented in \u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ed\u003c/b\u003e are the FTIR spectra of different samples. A very broad peak can be seen from around 3,600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 3,100 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the optimized FIN dry powder. This is likely due to the formation of intermolecular hydrogen bonding between CUR and PVP [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], which improved the stability of the nanoparticles. Regarding the \u003cem\u003ein vitro\u003c/em\u003e drug release, while the optimized FIN dry powder showed rapid and good redispersion (RdI\u0026thinsp;\u0026lt;\u0026thinsp;1.2) in simulated nasal fluid, neither FIN nor CUR were detectable by HPLC throughout the 3-hour experiment. The FIN or CUR in the physical mixture in the simulated nasal fluid also could not be detected. However, the release of FIN and CUR from the optimized FIN dry powder surpassed those of its physical mixture counterpart in an 80:20 (\u003cem\u003ev/v\u003c/em\u003e) solution of simulated nasal fluid and ethanol (\u003cb\u003eFig. S3\u003c/b\u003e). It is believed that no drug release occurred in the physical mixture due to its limited solubility, but for the nanosuspension reconstituted from the optimized FIN dry powder, most of the drugs were well encapsulated and protected within the nanoparticles. The effective encapsulation of drugs within the nanoparticles also renders the optimized dry powder less susceptible to nasal mucociliary clearance (typically occurring every 10\u0026ndash;20 minutes) [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], making it suitable for nose-to-brain drug delivery.\u003c/p\u003e \u003cp\u003eThe nasal deposition studies of the optimized FIN dry powder were conducted using two different nasal anatomical models at varying flow rates of 0, 7.5, and 15 L/min. The flow rates were selected based on the typical breathing patterns pertinent to clinical applications: a flow rate of 15 L/min mimics normal steady breathing [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], 7.5 L/min corresponds to slow inhalation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and 0 L/min represents an absence of breathing or a breath-holding scenario [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. All nasal models were connected to an NGI to simultaneously study powder deposition in the nose and the respiratory tract.\u003c/p\u003e \u003cp\u003eThe powders demonstrated excellent dispersibility upon actuation of the nasal device, with the nasal device fraction consistently below 5% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), except for the 3D-printed nasal cast at 0 L/min flow rate. These findings suggest that the nasal device is generally effective for administering powder formulations to the nasal cavity. The high deposition in the nasal device (~\u0026thinsp;20%) observed for the 3D-printed nasal cast at 0 L/min flow rate may be attributed to the powder backflow from the respiratory region of the cast to the exterior of the nasal device due to insufficient energy from the flow.\u003c/p\u003e \u003cp\u003eThe olfactory pathway is an important route for nose-to-brain delivery [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. In the 3D-printed nasal cast (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), the deposition fractions of optimized FIN dry powders in the olfactory region were 45.4% for 15 L/min, 45.2% for 7.5 L/min, and 48.5% for 0 L/min. Hence, as high as 45% of the optimized FIN dry powder can deposit in the olfactory region. In the AINI model (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), the deposition fractions of optimized FIN dry powder in the olfactory region were 8.6% for 15 L/min, 4.7% for 7.5 L/min, and 7.3% for 0 L/min. Aside from the olfactory region being the primary focus for nose-to-brain delivery, the highly vascularized respiratory region also warrants attention, as drugs can be absorbed in this area for systemic delivery. In addition, parts of the turbinate region innervated by the trigeminal nerve can transport the drug from the nose to the brain via the trigeminal pathway [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. In the 3D-printed nasal cast, the deposition fractions of optimized FIN dry powder in the respiratory region were 48.3% for 15 L/min, 48.5% for 7.5 L/min, and 29.2% for 0 L/min. In the AINI model, the deposition fractions of optimized FIN dry powder in the respiratory region (sum of nasopharynx, turbinates, and vestibule) were 62.3% for 15 L/min, 62.3% for 7.5 L/min, and 85.0% for 0 L/min. In detail, the deposition fractions of optimized FIN dry powder in the turbinate region were 25.6% for 15 L/min, 29.2% for 7.5 L/min, and 62.5% for 0 L/min.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe discrepancy in regional deposition profiles between the 3D-printed nasal cast and the AINI was not surprising and can be ascribed to the differences in the design and construction materials of these models. Since the olfactory region of the 3D-printed nasal cast has a larger surface area, the deposition fraction was higher in this model. Apart from the design, the 3D-printed nasal cast was made of polylactic acid, while the AINI model was fabricated from stainless steel. The adhesion capabilities, texture, and potential impact of surface charges on dry powders may exhibit variations. In the present study, the FIN nano-embedded powders displayed a greater propensity to adhere to plastic surfaces. Powders in the olfactory region of the AINI model may have unintentionally fallen into the respiratory region (especially during the disassembly of the AINI), resulting in a lower deposition fraction. The \u003cem\u003ein vitro\u003c/em\u003e-\u003cem\u003ein vivo\u003c/em\u003e correlation of drug deposition using these nasal models should be further investigated.\u003c/p\u003e \u003cp\u003eIt is worth noting that the inspiratory flow rate may vary among patients of different ages and disease states. Therefore, a holistic evaluation of how the inspiratory flow rate may influence the nasal deposition is desired. Perkušić et al. found that an increased inspiratory flow rate from 0 L/min to 60 L/min was associated with reduced drug deposition in the olfactory region [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], while Rigaut et al. found that flow rate (0, 15, and 60 L/min) was not a significant influencing factor for olfactory deposition [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. In this study, the deposition fraction of the olfactory region was independent of the flow rate for the optimized FIN powder formulation under different flow rates in two different nasal models (\u003cb\u003eFig. S4\u003c/b\u003e). The reason for this observation could be due to the restricted range of inspiratory flow rate (0\u0026ndash;15 L/min) studied. Besides, the manufacturer of the Unidose Nasal Spray Device (Aptar Pharma) has suggested that simultaneous actuation of the nasal device and inspiration is not required because the nasal device can generate sufficient air pressure for powder dispersion [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Hence, the optimized powder formulation is suitable for patients with different inspiratory flow rates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec44\" class=\"Section2\"\u003e \u003ch2\u003e3.4 \u003cem\u003eIn Vitro\u003c/em\u003e Cytotoxicity Studies of the Optimized FIN Nano-embedded Dry Powder Formulation\u003c/h2\u003e \u003cp\u003eThe cytotoxicity of FIN nanosuspension reconstituted from the optimized dry powder was evaluated in various cell lines. SH-SY5Y and PC 12 cells were used as neuronal cell models, while RPMI 2650 and Calu-3 were used as nasal epithelial cell models [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. In addition, the cytotoxicity of raw FIN and a physical mixture of the optimized powder formulation composition were also investigated for comparison purposes. The concentration range studied (2.5\u0026ndash;1,000 nM) was chosen based on literature reports and clinical pharmacokinetics of FIN [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. As presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the cell viability for all groups was around 100%, with no significant difference observed. This result indicates that the formulation has an acceptable \u003cem\u003ein vitro\u003c/em\u003e safety profile.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec45\" class=\"Section2\"\u003e \u003ch2\u003e3.5 \u003cem\u003eIn Vivo\u003c/em\u003e Neuroprotective Effects of the Optimized FIN Nano-embedded Dry Powder Formulation\u003c/h2\u003e \u003cp\u003eA schematic diagram of the study design of \u003cem\u003ein vivo\u003c/em\u003e neuroprotective effects is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea. FIN nanosuspension was reconstituted from the optimized dry powder and separately delivered to the mice with MCAO surgery via intravenous (IV) and intranasal (IN) administration. To assess the neuroprotective effect with greater clinical relevance, the tested drugs were administrated 30 minutes after stroke surgery. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb, the brain slices were stained with Nissl solutions on day 2 and the infarct regions were marked by the red line. The infarct size was significantly reduced to 1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 mm\u003csup\u003e2\u003c/sup\u003e in the IV-treated group compared with the untreated MCAO group (1.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 mm\u003csup\u003e3\u003c/sup\u003e). Remarkably, the infarct size was further reduced to 0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 mm\u003csup\u003e2\u003c/sup\u003e in the IN-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. To precisely measure the cerebral infarct size, we also employed the 9.4T animal magnetic resonance imaging system (MRI) 24 hours post-treatment. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea shows the mouse brain MRI images with clear cerebral infarction after MCAO surgery in the left hemispheres, with an average infarct volume of 2.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71 mm\u003csup\u003e3\u003c/sup\u003e. The IV-treated mice had a lower infarct volume of 2.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85 mm\u003csup\u003e3\u003c/sup\u003e, while the IN-treated group gave a significant reduction to 1.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57 mm\u003csup\u003e3\u003c/sup\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eThe neuroprotective effects were also evaluated using the adhesive removal test, balance beam test, rotarod test, and the Longa score. As expected, the time required for the mice to remove the adhesive tapes from their bilateral paws after MCAO surgery was significantly increased due to the neurological deficit after acute ischemia imposed by the MCAO surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea, b). However, the time required for MCAO mice with IV and IN treatment to remove the adhesive tapes decreased significantly compared to untreated MCAO mice on day 2 and day 3 (Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eb) after stroke surgery. Notably, the effect on the IN-treated group on day 3 was more significant than that of the IV-treated group, as evidenced by the smaller \u003cem\u003ep\u003c/em\u003e-value. Likewise, MCAO mice treated with reconstituted FIN nanosuspension intranasally required less average time to cross the beam compared to untreated MCAO mice and IV-treated MCAO mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ec and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. For the rotarod test, however, the time spent on the rod for treated groups was not statistically significant from that in the untreated group on day 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ee), possibly due to the sensitivity of the rotarod test for the stroke model [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Nevertheless, a statistically significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) was observed between the MCAO\u0026thinsp;+\u0026thinsp;IN FIN and MCAO groups on day 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ef). The Longa scores of mice in both treatment groups significantly decreased compared to the untreated MCAO group (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eg). Taken together, these results suggest that the IN administration outperformed IV administration in terms of alleviating neurological deficits after a stroke.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, a western blot analysis was performed on post-stroke mouse brains to understand the neuroprotective mechanism. Expression of pro-apoptotic proteins BAX and CC3, and the anti-apoptotic protein BCL-2 in the peri-infarct tissue of our MCAO stroke model were determined [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. This was due to reports that FIN could increase the expression of BCL-2 and decrease the expressions of CC3 and BAX [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e, administration of the reconstituted FIN dry powder for nanosuspension by IV and IN routes showed significant neuroprotective effects by reducing the expression of apoptosis proteins CC3 and BAX and increasing the expression of anti-apoptotic protein BCL-2. Of note, expression of BAX in the peri-infarct tissue was statistically significantly lower in IN-treated mice compared to that in IV-treated mice. BCL-2 expression was also numerically higher in IN-treated mice relative to IV-treated mice despite the lack of statistically significant differences. Nevertheless, these results highlight that IN delivery of FIN nanoparticles was more effective in reducing infarct size in the peri-infarct tissue than IV delivery.\u003c/p\u003e \u003cp\u003eIt is noteworthy that the neuroprotective effects could be achieved after single-dose IN administration. The non-invasive and convenient IN administration method also presents a unique treatment modality where FIN nanoparticles (in form of dry powder or reconstituted nanosuspension) can be timely administered by ambulance staff or caregivers to patients experiencing acute ischemic stroke symptoms before hospital admission. This strategy can attenuate neuronal injury in the hyperacute phase of ischemic stroke between stroke onset and in-hospital treatment procedures and promote post-stroke functional recovery. Whilst cardiovascular adverse effects such as bradycardia and atrioventricular blockade have been observed in patients on chronic oral FIN treatment for multiple sclerosis [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e], we do not anticipate significant safety issues arising from single-dose IN nanoparticle administration due to its excellent cytocompatibility after single-dose treatment. A constraint of the current study is that mice were administered IN with reconstituted FIN nanosuspensions instead of FIN nano-embedded powder as precise powder administration to the small nares of rodents is challenging. Nonetheless, this study provides proof-of-concept evidence of the neuroprotective effects by FIN nanoparticles. It is anticipated that the administration of nano-embedded powders would offer prolonged nasal retention and thus further enhance nose-to-brain FIN transport and neuroprotective efficacy. Studies are ongoing to devise an appropriate technique for administering powder into the nares of rodents and investigate the dose-response relationship and pharmacokinetics of intranasally administered FIN nano-embedded powder in rodent MCAO models. The ultimate goal is to identify an optimal powder dose that can serve as an effective and rapid neuroprotective therapy after an acute ischemic stroke.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eA FIN nano-embedded dry powder formulation for intranasal application was developed using a full factorial design of experiments. The optimized FIN nanosuspension had a particle size of 134.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 nm, a satisfactory PDI, and acceptable stability. The nanosuspension was then spray-dried into a nano-embedded microparticles dry powder with the aid of mannitol. The optimized dry powder exhibited excellent redispersibility (RdI\u0026thinsp;=\u0026thinsp;1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04) and good drug deposition in the olfactory region. The deposition fractions in the olfactory region were found to be independent of the nasal inspiratory flow rate, rendering it suitable for patients with different clinical conditions. It also had acceptable safety profiles in both nasal and brain cell models. Improved behavioral test results, reduced infarct volume, altered expressions of anti-apoptotic and pro-apoptosis proteins were observed following IN administration of the reconstituted FIN nanosuspension in a MCAO mouse model. This study demonstrates the neuroprotective effects of IN and IV FIN nanoparticles, with IN administration showing superiority for ischemic stroke management. Investigations into dose-response effects and pharmacokinetics of the FIN nano-embedded dry powder via IN and IV administrations are ongoing to further optimize its efficacy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAINI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlberta Idealised Nasal Inlet\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eATCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAmerican Type Cultural Collection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBAX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eB-cell lymphoma 2 associated X\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBCL-2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eB-cell lymphoma 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCC3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecleaved Caspase-3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCLT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003echolesterol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCQA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecritical quality attributes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCUR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecurcumin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edrug loading\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDLS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edynamic light scattering\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMEM/F12\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco\u0026rsquo;s Modified Eagle Medium/Nutrient Mixture F-12\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDSC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edifferential scanning calorimetry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDoE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDesign of Experiment\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eencapsulation efficiency\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEtOH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eethanol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eF-12K\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKaighn's Modification of Ham's F-12\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efetal bovine serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFIN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efingolimod\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFTIR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFourier-transform infrared spectroscopy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eintranasal\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eintravenous\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMCAO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emiddle cerebral artery occlusion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMinimum Essential Medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMeOH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emethanol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMIVM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emulti-inlet vortex mixer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMRI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emagnetic resonance imaging\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNGI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enext generation impactor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePDI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolydispersity index\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePVP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolyvinylpyrrolidone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePXRD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epowder x-ray diffractometry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRdI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eredispersibility index\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRe\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReynold number\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eS.D.\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003estandard deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etransmission electron microscopy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTFA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etrifiuoroacetic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTGA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ethermogravimetric analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUPW\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eultra-purified water\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthics approval\u003c/h2\u003e \u003cp\u003eAll animal studies followed the ethical policies and guidelines recommended by ARRIVE (Animal Research: Reporting of In Vivo Experiments) and National Research Council's Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of the Animal Experimentation of Jinan University (Approval No.: 20230901-0012).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was financially supported by the University of Hong Kong (Project Number: 104006626), the Health@InnoHK programme from the Innovation and Technology Commission, Hong Kong SAR government, the Guangdong Basic and Applied Basic Research Foundation, China (2023B1515120035, 2024A1515012035) and the Science and Technology Planning Project of Guangdong Province, China (2020A0505100045).\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003e \u003cb\u003eXinyue Zhang\u003c/b\u003e: Conceptualization, Methodology, Investigation, Formal Analysis, Writing \u0026ndash; Original Draft, Visualization. \u003cb\u003eGuangpu Su\u003c/b\u003e: Methodology, Investigation, Formal Analysis. \u003cb\u003eZitong Shao\u003c/b\u003e: Investigation, Formal Analysis. \u003cb\u003eHo Wan Chan\u003c/b\u003e: Methodology, Investigation. \u003cb\u003eSi Li\u003c/b\u003e: Investigation. \u003cb\u003eStephanie Chow\u003c/b\u003e: Investigation, Visualization. \u003cb\u003eChi Kwan Tsang\u003c/b\u003e: Methodology, Supervision, Funding acquisition, Writing \u0026ndash; Review \u0026amp; Editing. \u003cb\u003eShing Fung Chow\u003c/b\u003e: Conceptualization, Methodology, Formal Analysis, Resources, Supervision, Project administration, Funding acquisition. Writing \u0026ndash; Review \u0026amp; Editing.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors gratefully acknowledge the assistance of Mr. Hok Wai Lee from the Department of Pharmacology and Pharmacy at the University of Hong Kong in HPLC method development. SEM and TEM studies were conducted with the support of the Electron Microscope Unit at the University of Hong Kong.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKatan M, Luft A. Global Burden of Stroke. Semin Neurol. 2018;38(2):208\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y-L, et al. Discovery of phenylcarbamoyl xanthone derivatives as potent neuroprotective agents for treating ischemic stroke. Eur J Med Chem. 2023;251:115251.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSekerdag E, Solaroglu I, Gursoy-Ozdemir Y. 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Mult Scler. 2016;22(2):201\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStatements \u0026amp; Declarations\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"drug-delivery-and-translational-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ddtr","sideBox":"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ddtr/default.aspx","title":"Drug Delivery and Translational Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Nose-to-brain drug delivery, Neuroprotection, Ischemic stroke, Fingolimod, Nanoparticles, Particle engineering, Nasal powder","lastPublishedDoi":"10.21203/rs.3.rs-4715108/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4715108/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIschemic stroke is one of the major diseases causing varying degrees of dysfunction and disability worldwide. The current management of ischemic stroke poses significant challenges due to short therapeutic windows and limited efficacy, leading to a pressing need for novel neuroprotective treatment strategies. Previous studies have shown that fingolimod (FIN) is a promising neuroprotective drug. Here, we report the rational development of FIN nano-embedded nasal powders using full factorial design experiments, aiming to provide rapid neuroprotection after ischemic stroke. Flash nanoprecipitation was employed to produce FIN nanosuspensions with the aid of polyvinylpyrrolidone and cholesterol as stabilizers. The optimized nanosuspension was subsequently spray-dried into a dry powder, which exhibited excellent redispersibility (RdI = 1.09 ± 0.04) and satisfactory drug deposition in the olfactory region using a customized 3D-printed nasal cast and an Alberta Idealized Nasal Inlet model. The safety of the optimized FIN dry powder was confirmed in cytotoxicity studies with nasal and brain cells, while the neuroprotective effects were demonstrated by observed behavioral improvements and reduced cerebral infarct size in an established mouse stroke model. The neuroprotective effect was further evidenced by increased expression of anti-apoptotic protein BCL-2 and decreased expression of pro-apoptotic proteins CC3 and BAX in brain peri-infarct tissues. Our findings highlight the potential of nasal delivery of FIN nano-embedded dry powder as a rapid neuroprotective treatment strategy for acute ischemic stroke.\u003c/p\u003e","manuscriptTitle":"Rational Development of Fingolimod Nano-embedded Microparticles as Nose-to-Brain Neuroprotective Therapy for Ischemic Stroke","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-19 06:29:46","doi":"10.21203/rs.3.rs-4715108/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2024-09-06T09:59:38+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-07-28T06:05:43+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-22T19:41:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-11T06:44:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Drug Delivery and Translational Research","date":"2024-07-09T22:53:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"drug-delivery-and-translational-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ddtr","sideBox":"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ddtr/default.aspx","title":"Drug Delivery and Translational Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3d88c132-e796-4c8f-bd6c-7eae2c3ca1a5","owner":[],"postedDate":"August 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-04T16:26:50+00:00","versionOfRecord":{"articleIdentity":"rs-4715108","link":"https://doi.org/10.1007/s13346-024-01721-8","journal":{"identity":"drug-delivery-and-translational-research","isVorOnly":false,"title":"Drug Delivery and Translational Research"},"publishedOn":"2024-11-01 16:20:16","publishedOnDateReadable":"November 1st, 2024"},"versionCreatedAt":"2024-08-19 06:29:46","video":"","vorDoi":"10.1007/s13346-024-01721-8","vorDoiUrl":"https://doi.org/10.1007/s13346-024-01721-8","workflowStages":[]},"version":"v1","identity":"rs-4715108","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4715108","identity":"rs-4715108","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}