Polysorbate-80 coated Galantamine-Chitosan nanoparticles for enhanced safety and efficacy: Pharmacokinetics and Brain distribution in Rats

Polysorbate-80 coated Galantamine-Chitosan nanoparticles for enhanced safety and efficacy: Pharmacokinetics and Brain distribution in Rats | 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 Polysorbate-80 coated Galantamine-Chitosan nanoparticles for enhanced safety and efficacy: Pharmacokinetics and Brain distribution in Rats Tamilselvan Natarajan, Vijaya Raghavan Chellan, Habibur Rahman, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5308408/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In brain drug delivery, the BBB (Blood-Brain Barrier) has been a great barrier for various active pharmaceutical drugs. Hence, developing suitable drug delivery systems are need of the hour. However, the present study aimed to develop a polysorbate-80 (PS-80) coated galantamine (GAL) loaded chitosan nanoparticles (CS-NPs) with particles less than 200 nm to enhance the drug internalization in the brain. GAL-CS-NPs were prepared using the ionic gelation technique and optimized to obtain a particle size of less than 200nm and further coated wirh PS-80 to obtain polysorbate-80 coated galantamine loaded chitosan nanoparticles (PS-80-GAL-CS-NPs) The physiochemical properties of uncoated GAL-CS-NPs, PS-80 coated GAL-CS-NPs and the in-vitro evaluations, such as cytotoxicity and cellular internalization in SH-SY-5Y human neuroblastoma cell lines, were studied. The particle size of optimized PS-80-GAL-CS-NPs was observed to be 115±4 nm, and zeta potential was found to be 31.2±1.7 mV. While the drug entrapment efficiency was found to be 65.5±1.2 %. The in-vitro drug release of PS-80-GAL-CS-NPs was found to be 56.75±1.3 %. However, the cytotoxicity studies did not show any toxicity for the drug concentrations of 10 and 100 µg/mL. PS-80-GAL-CS-NPs facilitated time-dependent GAL uptake in SH-SY-5Y cell lines. Studies conducted in vivo in plasma revealed a steady release of GAL from PS-80-GAL-CS-NPs. The distribution PS-80- GAL-CS-NPs in the brain after oral administration at 1 st , 4 th and 8 th hr was found to be 1.7, 3.1 and 2.0 folds higher when compared to the uncoated NPs. Further, the histopathological study did not show any morphological changes in the rat brain. This study indicates that PS-80-GAL-CS-NPs might be a promising delivery method for Alzheimer’s Disease (AD) Alzheimer brain targeting blood-brain barrier galantamine nanoparticle neuroblastoma SH-SY5Y cell lines Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction AD disease is a neurologically degenerative disorder becoming an increasingly difficult challenge to families, the healthcare industry, and caregivers [1]. Even though a variety of marketed drugs are authorized for AD, it remains a significant area of unmet medical need due to limitations like higher doses required for therapeutic effect, limited bioavailability, and poor absorption in the brain [2]. Also, it has been reported as it has significant peripheral side effects owing to increased2 absorption by normal cells, and it has difficulty in penetration at blood brain barrier (BBB) [3]. Galantamine (GAL) is a competitive acetylcholinesterase inhibitor used for a broad spectrum of AD symptoms and authorized by the USA FDA (“Food and Drug Administration”) and the EMA (“European Medicines Agency”) [4]. Dual method of action on the cholinergic system, inhibiting acetylcholinesterase and modulating nicotinic acetylcholine receptors allosterically [5]. In recent research, it was shown that GAL could also inhibit Aβ aggregation as well as Aβ induction of neuronal apoptosis [6,7]. But the drug’s clinical utility is hindered by its poor retention in the CNS (central nervous system) and the complexity of transporting it around BBB [8,9]. Several therapeutic strategies have been employed to enhance CNS bioavailability [10]. RMT (“Receptor-Mediated Transcytosis”) or AMT (“Absorptive-Mediated Transcytosis”) through the cerebral endothelium is the outcome of medication combined with a particular brain transport targeted [11]. Biodegradable polymeric drug carriers like nanoparticles (NPs) and liposome composed of natural, synthetic, semi-synthetic polymers and lipids have been applied to the target brain [12]. Among these, polymeric NPs are promising carrier since it has the capacity to open the tight junctions of BBB. Also, it could prolong the drug release and protect the drug from enzymatic degradation [13]. It has been reported that NPs containing hydrophilic polymers have the additional advantage of longer blood circulation, which would enhance extravasation & passive targeting [14]. Numerous materials, including natural and synthetic polymers, are used as drug carriers; among them, the natural polysaccharide chitosan (CS) has gained popularity due to its outstanding set of physical and biological properties [15]. CS is a natural polysaccharide composed of copolymers of glucosamine and N-acetylglycosamine. Recently CS has arisen as a promising alternative for improving the transport of such macromolecules across biological barriers for drug delivery and biomedical applications [16]. It was reported that the overcoating of the nanoparticles with polysorbates-80 (PS-80) capable of transporting drug across BBB the precise mechanism is not well established [17]. The overcoating of the particles lead to adsorption of apolipoprotein E from blood plasma foiiowed by receptor mediated endocytosis in then present investigation, we attempted to asses polysorbate − 80 coated crosslinked with chitosan nanoparticles for brain targeting of GAL. The in-vivo pharmacokinetics and brain distribution analyses were performed in rats to calculate the efficacy of PS-80-coated GAL-CS-NPs in the brain 2. Results 2.1. Preparation and optimization of NPs A straightforward, scaled-up version of the previously reported ionotropic gelation process was used to create CS-NPs [20]. In order to create NPs, cationic polyelectrolyte polymer CS is utilized. The NPs were created by regulating the gelation’s interaction with the polyanion TPP, which decreased the solubility of CS in water. The NPs were created by regulating the gelation’s interaction with the polyanion TPP, which decreased the solubility of CS in water. The rate-limiting step, which regulates the size and size distribution of NPs, is the CS/TPP ratio [21]. In order to obtain NPs under 200 nm, various ratios of CS/TPP were used in the formulation of NPs. To attain the ideal CS and TPP ratio, several concentrations of GAL-loaded CS and TPP NPs were produced via the ionic interaction of positively charged CS and negatively charged TPP. 2.2. Physiochemical characterization of NPs The zeta potential, PDI, mean particle size, and drug loading of uncoated GAL-CS-NPs were studied (Table 1 ). The mean particle size and PDI varied from 102 ± 4 to 4852 ± 8 nm and 0.28 ± 0.07 to 0.91 ± 0.31, respectively, depending upon the concentration of TPP and CS. The zeta potential was in the range of 31.2 ± 1.7 to 40.0 ± 1.5 mV, and the drug loading varied from 42.3 ± 1.6 to 70.6 ± 1.5%. Later PS-80 was used to modify the surface of GAL-CS-NPs. Considering the fact that polymeric NPs modified by PS-80 were taken up rapidly by brain micro vessel endothelial cells, an attempt was made to enhance the uptake of GAL in the brain by modifying the surface of NPs using PS-80. The mean particle size of PS-80 coated GAL-CS-NPs containing 0.6 and 0.2% of CS was found to be 98 ± 4 and 126 ± 3 nm, respectively. Zeta potentials of 31.2 ± 1.7 and 40.0 ± 1.5 mV was discovered. After the NPs were coated with PS-80, there was a modest increase in particle size and a decrease in zeta potential owing to obscuring the NPs’ surface from the additional layer of PS-80 coating (Table 2 ). AFM and TEM were also used to characterize the GAL-CS-NPs. The acquired data indicates that individual particle sizes were in the 20–25 nm range. Because CS-NP has a high swelling capacity, the diameter of the particles assessed using the dynamic light scattering (DLS) approach was larger than the size calculated using TEM and AFM. The Zetasizer evaluates the hydrodynamic diameter based on how a particle diffuses inside a fluid. The particle’s surface structure, as well as the particle’s size, determine the hydrodynamic diameter. The GAL-CS-NPs 3 formulation with the small particle size and zeta potential value of greater than + 30 mV, which indicates the stability of NPs was selected as the optimized formulation. The external morphology of the optimized uncoated NPs was studied using TEM and AFM (Fig. 1 ). The TEM pictures demonstrated that the NPs were spherical in form without any aggregates of particles with a size range of 20 nm scale (Fig. 1 A), and it was further supported by the AFM image (Fig. 1 C&D), which revealed particles with a smooth surface devoid of any apparent pinholes or fractures. SAED of drug-loaded NPs in TEM revealed the amorphous state of GAL in CS-NPs. The SAED image clearly demonstrated the absence of a ring pattern owing to the presence of GAL in amorphous from (Fig. 1 B). This investigation provides proof that the CS-NPs’ integrated GAL was an amorphous substance. Table 1 Composition of CS-NPs, particle size, PDI, zeta potential and drug loading. Formulations CS (1%) TPP (%) Size (nm) PDI Zeta Potential (mV) Entrapment efficiency (%) GAL-CS-NPs 1 0.2 0.2 2628 ± 4 0.49 ± 0.03 6 ± 1 46.9 ± 1.8 GAL-CS-NPs 2 0.4 0.2 2444 ± 6 0.91 ± 0.31 12.6 ± 1.2 55.5 ± 1.1 GAL-CS-NPs 3 0.6 0.2 98 ± 4 0.35 ± 0.05 40.0 ± 1.5 65.5 ± 1.2 GAL-CS-NPs 4 0.2 0.4 126 ± 3 0.34 ± 0.07 31.2 ± 1.7 70.6 ± 1.5 GAL-CS-NPs 5 0.4 0.4 2574 ± 2 0.31 ± 0.04 5.7 ± 1.5 52.6 ± 2.05 GAL-CS-NPs 6 0.6 0.4 2953 ± 3 0.28 ± 0.07 4.6 ± 1 42.3 ± 1.6 GAL-CS-NPs 7 0.2 0.6 4731 ± 2 0.42 ± 0.05 5 ± 1.6 47.8 ± 1.4 GAL-CS-NPs 8 0.4 0.6 2852 ± 8 0.56 ± 0.07 4.8 ± 1.7 55.06 ± 1.9 GAL-CS-NPs 9 0.6 0.6 2764 ± 6 0.84 ± 0.08 3.2 ± 1.2 43.2 ± 1.7 Table 2 Particle size and zeta potential of GAL-CS-NPs after PS-80 coating. Formulation code Size (nm) PDI Zeta Potential (mv) PS-80-GAL-CS-NPs 3 115 ± 4 0.69 ± 0.03 31.9 ± 1.6 PS-80-GAL-CS-NPs 4 126 ± 3 0.15 ± 0.31 26 ± 1.8 The crystalline character of the NPs was investigated using DSC and SAED investigations. The thermograms for CS, GAL, physical mixture, and GAL-CS-NPs were developed (Fig. 2 ). The broad endothermic peak of pure GAL at 271.77 ºC was absent in the thermogram of GAL-CS-NPs, indicating the absence of crystalline material in the NPs. The absence of a peak might be due to the inhibition of crystallization of GAL by polymer during NPs preparation. This leads to the conclusion that the medicine included in the NPs was in an amorphous, molecular dispersion, solid solution, or disordered crystalline inside the matrix of the polymer. 2.3. In-vitro drug release study In-vitro release behavior of the uncoated GAL-loaded CS-NPs was examined in phosphate buffer (pH 6.5). The release profile demonstrated the cumulative drug release for the optimized formulations GAL-CS-NPs 3 and GAL-CS-NPs 4 (Fig. 5 A). The release of drug from drug-loaded NPs varied from 56.75 ± 1.3 to 65.2 ± 0.7% over a period of 24 hrs. It was apparent from the release profile that the NPs showed an initial burst release around 17.2 ± 1.8 and 16.3 ± 2.8% at the end of 1st hr and followed by a slow-release pattern of the drug was found to be 56.75 ± 1.3 and 65.2 ± 0.7%. The initial burst of GAL release may have been caused by adsorbed GAL on the NP surface, and the entrapped GAL slowly released from the NPs after that. The release data were fitted to the Korsmeyer-Peppas model, as shown by higher regression coefficients (r2-values), which were determined to be 0.8876 and 0.8975 for GAL-CS-NPs 3 and GAL-CS-NPs 4, respectively, in order to study the manner of drug release from CS-NPs. The obtained ‘n’ values 0.290 and 0.324 suggested that the drug releases followed the Fickian diffusion mechanism. 2.4. Storage stability studies The appearance of PS-80-GAL-CS-NPs was found to be pale white powder during the lyophilization process, and, there were no changes in appearance even after 6th month of storage at room temperature. The particle size of PS-80-GAL-CS-NPs was found to be 116 ± 4 on the initial day of preparation. After storage for 6 months, the size of particles was slightly increased in size as 125 ± 5, 138 ± 4, 149 ± 9 and 154 ± 6 nm in 1st, 2nd, 4th, and 6th months, respectively (Table 3 ). The drug entrapment efficiency also was found to be stable at 64.0 ± 0.6, 65.2 ± 0.2, 64.1 ± 0.8 and 66.4 ± 0.5 at 1st, 2nd, 4th and 6th months, respectively. Table 3 Stability studies of PS-80 coated GAL loaded CS-NPs. Temperature Evaluation parameter Observation (months) 0 1 2 4 6 24 o C (RH = 65%) Physical appearance Pale white powder No change No change No change No change Particle size (nm) 116 ± 4 125 ± 5 138 ± 4 149 ± 9 154 ± 6 Entrapment efficiency (%) 65.1 ± 0.8 64.0 ± 0.6 65.2 ± 0.2 64.1 ± 0.8 66.4 ± 0.5 2.5. Stability of drug in SGF The stability of GAL in SGF was examined. It was found that the percentage of drug remaining was found to be 72 ± 2.5 and 75.3 ± 1.1%. The obtained result shows that the drug was protected from gastric enzymatic degradation for up to 3 hrs. It could be because PS-80 has lengthy chains with dense surfaces that act as a protective brush to stop the medicine from degrading. 2.6. In-vitro cell study 2.6.1. MTT Assay Cytotoxicity of uncoated GAL-NPs and PS-80 coated GAL-NPs was evaluated for screening novel compounds for neurotoxicity properties. For 12 and 24 h, the cells were treated with drug solution, uncoated CS-NPs, & CS-NPs coated with PS-80 at concentrations of 10 and 100 g/mL. The data showed that neither uncoated nor PS-80 coated GAL loaded NPs were found to be non-toxic on these SH-SY-5Y cells, even at the higher concentration of the formulation (Fig. 3 ). The cell viability was above 90% for all assays. 2.6.2. Cellular uptake study Fluorescence microscopy was used to examine the capacity of NPs-Rho loaded CS-NPs to be internally incorporated into SH-SY5Y human neuroblastoma cells [23]. The cellular uptake of uncoated Rho-CS-NPs was studied at the 1st and 4th hr. (Fig. 4 A&B). At 1st hr., the uptake of Rho on the cells was not visible, and after a few hrs., the uptake of Rho was seen in the surrounding of the cell membrane. With regard to Rho-NP coated with PS-80, the internalization of Rho was clearly seen inside the membranes after 1st and 4th hr. incubation (Fig. 4 C). The internalization of Rho was also seen in the entire membrane’s cytoplasm (Fig. 4 D). The internalization of Rho-PS-80-CS-NPs was higher with time when compared to the uncoated and free Rho. Positively charged CS and negatively charged cell membranes had strong ionic contact, which led to increased internalization. Further, the other possibility of internalization happened by the presence of PS-80 on the surface of NPs which got adsorbed on the apolipoproteins present on the cell surface through receptor-mediated endocytosis. Hence, NPs may be an effective and safe delivery systems for the oral administration of GAL. 2.7. In-vivo studies 2.7.1. Pharmacokinetic studies Wister male rats were used for in-vivo pharmacokinetics and tissue distribution experiments. Following oral administration of GAL solution, GAL-NPs, and GNP-PS-80, the plasma and tissue concentration of GAL at various time points was measured. With the aid of WinNonlin, the pharmacokinetic parameters were computed using non-compartmental analysis (version 5.1). The pharmacokinetic parameters such as Cmax, tmax, AUC0- 24, t1/2, and MRT (hr) were analyzed. Different formulations’ plasma concentration vs. time profiles were shown. Rats who were given GAL-NPs had a greater plasma concentration of GAL than rats that were given free GAL (Fig. 5 ). The Cmax of GAL after administration with GAL-NPs was significantly higher than the Cmax obtained after administration of GAL solution (P < 0.05), and a 5.03-fold increase was observed. A delayed Tmax reveals a prolonged release pattern of GAL-CS-NPs, and a rise in Cmax following treatment via NPs indicates an increase in the quantity of GAL that has entered the systemic circulation. This shows that GAL-NPs improve absorption by lowering the first-pass metabolism, resulting in enhanced medication availability for extended periods of time. GAL solution and GAL-NPs were found to have AUCs of 1064.12 and 6877.7 ng/mL, respectively. When given as NPs at 24 h, more GAL was seen in the plasma than in the GAL solution, indicating a sustained release pattern for CS-NPs. When compared to the plasma concentration profile of GAL-NPs, the plasma drug concentration profile obtained after administering PS-80 coated NPs showed a lower amount of GAL in blood, indicating that the PS-80 coating protected the release of drug in the plasma, enhancing the presence of GAL in the brain.[26] 2.7.2. Brain distribution studies The concentration of GAL in the brain was estimated by HPLC. The concentration of GAL (ng/tissue) was achieved in the brain at different time points after oral administration of GAL solution, GAL-NPs and GAL-NP-PS-80 at 1, 4 and 8th hrs post-administration (Fig. 5 ). The representing chromatogram of GAL concentration from GNP-PS-80 in the brain at different time points 1, 4 and 8 hrs are recorded (Fig. 5 ). The NPs formulation facilitated the improved uptake of the drug and availability in both plasma and brain tissue. The drug concentration was found to be 32.7 ± 3.2, 28.6 ± 2.1 and 17.9 ± 8.5 ng/tissue after 1, 4 and 8th hrs. The plain GAL solution showed a decrease in the availability of the drug. When administered as NPs, the drug concentration showed a little increase in the brain, indicating the sustained release of CS-NPs. The concentration of NPs was found to be 226.1 ± 1.3, 114.4 ± 5.3, and 110.8 ± 5.9 ng/tissue in the case of PS-80 coated NPs, showing enhanced drug availability in the brain at all time points. PS-80 coating indicated the enhanced uptake of GAL in the brain through receptor-mediated endocytosis and showed the importance of surface properties in the biodistribution of NPs. Additionally, adsorptive-mediated transcytosis induced by ionic interaction between the negatively charged membrane surface and the positive charge of NPs facilitated an increased permeation across BBB.[22] The ratio of drug concentration in the brain to that in plasma is often used as a targeting indicator. Rats were used to study the ratio of brain to plasma concentration of the GAL formulations (GAL-solution, GAL-NPs, and GNP-PS-80) at various time points after oral treatment (Fig. 5 ). The findings show that the GNP-PS-80 formulation’s brain-to-plasma ratio was consistently larger than one at all time periods (1, 4 and 8th hrs.). The preferential localization of GAL in the brain for CS-NPs coated with PS-80 when compared with the GAL-solution and uncoated GAL-NPs can be attributed to endocytosis. These results indicate that the PS-80 coated CS-NPs after oral administration enhanced the delivery of GAL to the brain. 2.7.3. Histopathological studies After administering NPs orally to the cerebral area, photomicrographs of brain slices were examined for morphological alterations, including inflammation of neurons. The GAL-Solution treated GAL-NPs and PS-80 coated GAL-NPs did not exhibit any morphological abnormalities, such as vacuolization/spongiosis and neuronal degeneration in the morphological sections, which mimic brain sections of normal nerve cells (Fig. 6 ). Therefore, it is evident that the polymeric NPs with PS-80 coated were safe for the brain [24] 3. Materials and Methods 3.1. Materials Chitosan low molecular weight was purchased from Sigma-Aldrich, USA, sodium tripolyphosphate (TPP) was purchased from Loba Cheme, India. The Glacial acetic acid was obtained from Fisher Scientific, USA. Galantamine hydrobromide was obtained as a gift sample by Alembic, India. Dialysis membrane with a molecular weight cut off 12,000–14,000 Daltons was purchased from HI Media Laboratories, India. 3.2. Synthesis of nanoparticles The ionic gelation process was used to create GLA-CS-NPs [18]. Determinate weights of CS (0.2, 0.4 and 0.6%) were dissolved in glacial acetic acid 1 percent (v/v). Aqueous sodium tri poly phosphate (TPP) solution was added in a dropwise manner at concentrations of 0.2, 0.4, and 0.6 percent after GAL was added to CS while being constantly magnetically stirred. For 30 min, the mixture was continuously stirred with magnets. To eliminate excessive levels of TPP and unencapsulated free GAL, the suspension of NPs was centrifuged at 13,000rpm for 30 min at 4◦C using an Ultracentrifuge. The pellets have been dispersed within deionized water. Finally, NPs have been lyophilized with a freeze dryer for 24 hrs. (Lyodel, India) in preparation for powder storage. To prepare fluorescently loaded CS-NPs, Rhodamine-6G was dissolved in ethanol (20 mg/mL) and further combined in 10 mL of CS solution (0.6%) and cross-linking with 0.2percent w/v TPP solution at room temperature with constant stirring for 30 min at 1,000 rpm. The NPs have been centrifuged at 13,000rpm at 4ºC for 30min, and the pellet was separated and stored until further study. 3.3. Coating of GLA-CS-NPs The NPs coating was conducted as per the procedure defined by [19]. The freeze-dried drug-loaded NPs (20 mg per ml) were resuspended in PBS (“Phosphate Buffered Saline”) under constant stirring at 50 rpm (Remi Motors, India). Then PS-80 was added to have a final solution of 1 percent (v/v) mixture and was incubated for 30min. The freeze-dried NPs were subjected to zeta potential, particle size, along with drug loading characterizations. 3.4. Physiochemical characterization of GAL-CS-NPs 3.4.1. Zeta potential and particle size measurement The average hydrodynamic diameter as well as PDI (“Polydispersity Index”) of the formulated NPs were examined by Zetasizer Nano ZS90 (Malvern Instruments Ltd., United Kingdoms), using DLS (“Dynamic Light Scattering”) technique. For the purpose of measuring particle size, a 1ml sample of NPs dispersion was deposited in disposable cuvettes. Each experiment was carried out three times. The Malvern Zetasizer (Nano ZS90, Malvern Instruments) was used to evaluate electrophoretic mobility (zeta potential) at a temperature of 25ºC. Double-distilled water was used to dilute the samples. 3.4.2. TEM (Transmission Electron Microscopic) analysis The prepared NPs’ morphology was assessed using a TEM fitted with a SAED (Selected Area Electron Diffraction) Pattern. An NPs suspension drop was placed on a copper grid, and the scanning was conducted at 80kv (“JOEL JEM 2100, Japan”). The morphology of the copper grid was examined while it was in the vacuum chamber of a TEM with the grid-connected to a sample holder. 3.4.3. Atomic force microscopic (AFM) determination Under typical air circumstances, an AFM (“Nova NTEGRA prima, Russia”) was used to observe the surface characteristics of drug-loaded NPs. The scan rate was set at 2 Hz. The samples were placed on glass slides after being diluted ten times with distilled water and vacuum dried at 25°C for 24 hrs. AFM image analysis software (“Multimode scanning probe microscope”) was used to estimate height. 3.4.4. Drug encapsulation determination The drug EE (Encapsulation Efficiency) of Gal-CS-NPs was examined as per the approach depicted (Vila et al., 2002 ). Each batch included 50 mg of drug-loaded CS-NPs, which were digested for 24 h in a 20 mL solution of 0.1 N HCl and 1:1 v/v ethanol. The particles have been separated using centrifugation at 13,000rpm, and the drug concentration in the supernatant was examined using Ultraviolet spectrophotometry at 289nm. % EE=(Weight of total drug-Weight of free drug)/(Weight of total drug)×100 (1) 3.4.5. Differential scanning calorimetric (DSC) analysis GAL’s physical state inside the NPs was described by DSC. The thermogram of pure GAL, CS, physical mixture and GAL-CS-NPs was conducted using DSC. A standard aluminum pan was utilized to contain the sample (5mg), which was then crimped as well as heated at a rate of 10ºC/in from 4 to 450ºC while nitrogen was being purged (20 mL/ min). The empty sealed aluminum pan was employed as a reference. 3.5. In-vitro drug release A revised dialysis approach was applied to assess the in-vitro release of GAL-CS-NPs (Hu et al., 2003 ). A dialysis bag was filled with the NP’s suspension, which contained 2 mL or 2 mg of GAL. Both the ends of the dialysis bag were tied using membrane clips and placed into 20mL of “phosphate buffer” (0.1 M, pH 6.5), kept at 37ºC with continuous magnetic stirring. Aliquots from the releasing medium were taken out at predetermined times and replaced with the equivalent volume of phosphate buffer. GAL at 289 nm was measured spectrophotometrically in the sample. 3.6. In-vitro release kinetics DDSolver was used to calculate the drug release data, and the resulting data were fitted to the Korsmeyer-Peppas exponential equation to determine the mechanism of drug release, in which Q indicates the proportion of the drug released at time t and k denotes a constant incorporating the structural and geometric properties of the device under examination (Zhang et al., 2010 ). The mode of drug transport from the dosage form is significantly indicated by the diffusional exponent 𝑛. 3.7. Storage stability studies The storage stability of PS-80-GAL-CS-NPs was examined according to ICH (“International Conference on Harmonization”) recommendations. The formulations were stored at room temperature (24oC, RH = 65%) in the dark for six months. The physical appearance, average particle size, and entrapment efficacy of the stored formulations were examined during the 1st, 2nd, 4th and 6th months to determine their stability. 3.8. Stability of drug in SGF (Stimulated Gastric Fluid) The GAL stability was assessed in SGF, prepared as per “British Pharmacopoeia” (BP). In a nutshell, 80mL of 1M HCl was used to dissolve 2g of NaCl and 3.2g of pepsin before the volume was increased to 1000mL and the pH was set to 1.2. Typically, 15 mL of SGF was used to suspend 50 mg of every formulation, and the mixture was then put into screw-capped tubes. The water shaker bath was maintained at 37°C with the tubes. The suspensions have been centrifuged at 13,000 rpm for 40 min after 3 h of incubation in SGF, and the supernatants were discarded. The sediment was disseminated in the solvent, and the drug concentration was measured using UV light at 289 nm. 3.9. In-vitro cell studies 3.9.1. MTT assay National Center for Cell Science (NCCS), Pune, provided the human SH-SY5Y cells. Cells were cultured in MEM with non-essential amino acids, ham F-12, 10 percent fetal bovine serum, 2 mmol/L L-glutamine, penicillin (100 U/mL), and streptomycin (100µg/mL ) in a humid atmosphere at 37°C and 5 percent CO2. Every week, the medium was changed twice. Cell viability was evaluated with the “3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide” (MTT) test. SH-SY5Y cells were planted at a density of 1×104 cells per well in 96-well plates for 24 h under a 10 percent serum condition. Cells were kept in serum-free media for 16 h after the serum concentration was gradually reduced (5%) over a period of days. A normal control group, a placebo, and three different formulations of galantamine at doses of 10 &100 µg each were divided among the wells in triplicate. Cells were exposed to drug formulations for 24 h. MTT was produced at a concentration of 5 mg/mL, and 20µL of the MTT solution was applied to each well before being incubated for four hours. After the MTT had been incubated, the medium containing the MTT was taken out, and each well-received 50µL of DMSO was to dissolve the formazan crystals. At 570 nm, optical density was investigated. In comparison to the control, percentage toxicity was calculated. 3.9.2. Cellular uptake study Confluent SH-SY-5Y was used in the qualitative investigation. Human neuroblastoma cells were grown in MEM with non-essential amino acids, ham F-12, 10% fetal bovine serum, 2 mmol/L L-glutamine, penicillin (100 U/mL), and streptomycin (100 µg/mL) and kept in a humid atmosphere at 37°C and 5% CO2. Every week, the medium was changed twice. SH-SY-5Y cells were seeded in 96-well plates at a density of 1×104 cells per well, and they were subsequently incubated at 37 C with rhodamine-6 (Rho) loaded NPs. The cells were examined using fluorescence microscopy after being rinsed 3 times with cold PBS. 3.10. Bio-analytical method development The concentration of GAL was quantified using HPLC (“High-Performance Liquid Chromatography”) (waters 2489) equipped with a UV detector at 289 nm with a reversed-phase column (Gemini C18, 5µ,50x30mm Phenomenex). The mobile phase consists of 0.01M ammonium acetate buffer (pH 3) and ACN (94:6 v/v) at the flow rate of 0.5ml/minute. The extraction of GAL from tissue was conducted using liquid–a liquid extraction technique. Rat tissue homogenate (100µL) and 20µL of a solution containing 1 M sodium hydroxide were mixed and vortexed for 60 s. This mixture received 0.7 mL of tetra butyl methyl ether, which was then centrifuged at 10,000 rpm for 5 min. Following separation, the organic layer was exposed to a nitrogen gas stream while being enclosed by a turbo vap LV (Biotage USA) at 40°C. Finally, 20µL of the reconstituted mobile phase was used for HPLC analysis after the dried residue was re-formed with 100µL of the mobile phase. 3.11. In-vivo evaluations 3.11.1. Pharmacokinetic study The in-vivo pharmacokinetic tests were conducted on male Wistar rats weighing 200–250g. PSG Institutional Animal Ethics Committee (Proposal No. 197/2013/IAEC), located in Coimbatore, examined, and approved the protocol for the animal trials. The animals were housed in a well-ventilated animal home with a sterile paddy husk cage, a regular diurnal cycle, and full access to food and water. The night before the experiment, the animals were given permission to fast. Three groups of six animals each were formed from the total number of animals. The animals were given a dosage of 1 mg/kg orally using a gastric lavage needle. GAL solution was given to group 1 suspended in PBS 7.4, GAL-NPs were given to group 2 uncoated, and PS-80 coated GAL-NPs were given to group 3. At 60, 120, 240, 480, 720, and 1440 min after treatment, retro-orbital venous plexus punctures were used to collect the blood samples (500 L) into microcentrifuge tubes containing sodium citrate. The blood samples were centrifuged at 4,000 rpm for ten mins. For further studies, the separated plasma was stored at a temperature of − 20°C 3.11.2. Brain distribution study The Wister rats used in the tissue distribution assays weighed between 200 and 250 g. Three groups of six rats each were created from the group of rats. The drug was administered through the oral route. Groups 2 and 3 each got uncoated GAL-NPs at a dosage of 1 mg/kg, whereas Group 1 received a GAL solution. Three animals from each group were sacrificed after the first, fourth, and eighth hours of treatment. The brain’s target tissue was then removed and homogenized with ice-cold PBS 7.4 in a tissue homogenizer (Remi) (Gaur et al., 2000 ). HPLC, in conjunction with a UV detector, was applied to assessed the content of drug in the organs. 3.11.3. Histopathological analysis On Wister rats, a histopathological study was done. After 24 h, animals were slaughtered, and the brains were taken out. The brain tissue was embedded in paraffin after being treated in a 10% formaldehyde solution. 7 mm thick paraffin slices were created for microscopy research. Finally, after being submerged in water, the tissues were stained with hematoxylin and eosin for ten minutes at 60°C. The discolored areas were improved for dehydration using various grades of alcohol after being rinsed in running water to eliminate the surplus stain. Finally, the slides were made permanent by clearing them with xylene and mounting them with DPX. The slices were examined for the presence of neuronal degeneration, lipifuscin deposits, and vacuolization/spongiosis 4. Conclusions By using a modified ionic gelation technique and optimizing the particle size > 200 nm, PS-80 coated GAL-loaded CS-NPs were created, increasing their absorption in the brain. The pharmacokinetic data indicate a sustained release profile of GAL from CS-NPs when compared to GAL-Solution. Distribution studies to the brain proved that PS-80 coated GAL-NPs enhanced the drug availability in the brain for up to 8 hrs when compared with the uncoated NPs after oral administration. Therefore, the polymeric NPs developed after surface coating with PS-80 represent a promising approach to overcoming the BBB issues for the brain delivery of drugs to treat other CNS disorders. Declarations Author Contributions: Conceptualization: T.N, and V.R.C; Data collection and curing: T.N A.K, H.R, P.K.G and S.K; Primary Draft: T.N,V.R.C, K.A and K.S; Software: T.N, H.R, K.S and A.K; Final Draft: T.N, V.R.C, H.R, P.K.G, K.S, A.K Funding: The authors are also thankful to Indian Council of Medical Research (ICMR), New Delhi for the research grant titled “Development of nanoparticleculate drug delivery systems to treat Alzheimer’s disease” Ref No: 35/36/2010/Nano-BMS. Institutional Review Board Statement: PSG Institutional Animal Ethics Committee (Proposal No. 197/2013/IAEC), located in Coimbatore, examined, and approved the protocol for the animal trials. Informed Consent Statement: Not Applicable Data Availability Statement: Not Applicable Acknowledgments: The authors are thankful to PSG College of Pharamcy, Coimbatore for providing facilities to carry out the work Conflicts of Interest: The authors declare no conflict of interest References Kumar, A.; Sidhu, J.; Goyal, A. Alzheimer Disease. In StatPearls; StatPearls Publishing: Treasure Island, FL, USA, 2020. https://www.ncbi.nlm.nih.gov/books/NBK499922 Dong, X., Current Strategies for Brain Drug Delivery. Theranostics.2018.8,1481–1493. https://doi.org/10.7150/thno.21254 Zhao, D.; Yu, S.; Sun, B,; Gao, S, ; Guo, S, ; Zhao, K., 2018. Biomedical Applications of Chitosan and Its Derivative Nanoparticles. Polymers (Basel). 10, 462. https://doi.org/10.3390/polym10040462 Rubin, L.L,; Staddon, J.M,;. The cell biology of the blood-brain barrier. Annu. Rev. Neurosci. 1999, 22, 11–28. https://doi.org/10.1146/annurev.neuro. Lilienfeld, S. 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Sci, 2002. 6, 319–327. https://doi.org/10.1016/S1359-0286(02)00117-1 Hu, Y., Jiang, X., Ding, Y., Zhang, L., Yang, C., Zhang, J., Chen, J., Yang, Y., Preparation, and drug release behaviors of nimodipine-loaded poly(caprolactone)–poly (ethylene oxide)–polylactide amphiphilic copolymer nanoparticles. Biomaterials, 2003, 24, 2395–2404. https://doi.org/10.1016/S0142-9612(03)00021-8 Liu, Z., Jiao, Y., Wang, Y., Zhou, C., Zhang, Z., Polysaccharides-based nanoparticles as drug delivery systems. Adv. Drug Deliv. Rev. 2008, 60, 1650–1662. https://doi.org/10.1016/j.addr.2008.09.001 Genta, I,; Perugini, P,; Conti, B,; Pavanetto, F., A multiple emulsion method to entrap a lipophilic compound into chitosan microspheres. Int. J. Pharm, 1997. 152, 237–246. https://doi.org/10.1016/S0378-5173(97)00096-3 Arya, G,; Vandana, M,; Acharya, S;, Sahoo, S.K. Enhanced antiproliferative activity of Herceptin (HER2)-conjugated gemcitabine-loaded chitosan nanoparticle in pancreatic cancer therapy. Nanomedicine Nanotechnology, Biol. Med., 2011 7, 859–870. https://doi.org/10.1016/j.nano.2011.03.009 Ramge, P,; Unger, R.E,; Oltrogge, J.B,; Zenker, D,; Begley, D,; Kreuter, J,; Von Briesen, H.,. Polysorbate-80 coating enhances uptake of polybutylcyanoacrylate (PBCA)-nanoparticles by human and bovine primary brain capillary endothelial cells. Eur. J. Neurosci. 2000, 12, 1931–1940. https://doi.org/10.1046/j.1460-9568.2000.00078.x Vila, A,; Sánchez, A,; Tobı́o, M,; Calvo, P,; Alonso, M.J., Design of biodegradable particles for protein delivery. J. Control. Release 2002, 78, 15–24. https://doi.org/10.1016/S0168-3659(01)00486-2 Zhang, Y,; Huo, M,; Zhou, J,; Zou, A,; Li, W., Yao, C,; Xie, S., DDSolver: An Add-In Program for Modeling and Comparison of Drug Dissolution Profiles. AAPS J. 2010, 12, 263–271. https://doi.org/10.1208/s12248-010-9185-1 Prabhakar, K,; Afzal, S.M,; Kumar, P.U,; Rajanna, A,; Kishan, V. Brain delivery of transferrin coupled indinavir submicron lipid emulsions—Pharmacokinetics and tissue distribution. Colloids Surfaces B Biointerfaces., 2011, 86, 305–313. https://doi.org/10.1016/j.colsurfb.2011.04.013 Gaur, U,; Sahoo, S.K,; De, T.K,; Ghosh, P.C,; Maitra, A,; Ghosh, P.. Biodistribution of fluoresceinated dextran using novel nanoparticles evading reticuloendothelial system. Int. J. Pharm, 2000. 202, 1–10. https://doi.org/10.1016/S0378-5173(99)00447-0 Kakkar, V,; Kaur, I.P., Evaluating potential of curcumin loaded solid lipid nanoparticles in aluminum induced behavioral, biochemical and histopathological alterations in mice brain. Food Chem. Toxicol, 2011. 49, 2906–2913. https://doi.org/10.1016/j.fct.2011.08.006 Janes, K.A,; Calvo, P,; Alonso, M.J., Polysaccharide colloidal particles as delivery systems for macromolecules. Adv. Drug Deliv. Rev, 2001. 47, 83–97. https://doi.org/10.1016/S0169-409X(00)00123-X Ying, X,; Wen, H,; Lu, W.-.;, Du, J,; Guo, J,; Tian, W,; Men, Y,; Zhang, Y,; Li, R.-J., Yang, T.-Y,; Shang, D.-W,; Lou, J.-N,; Zhang, L.-R,; Zhang, Q. Dual-targeting daunorubicin liposomes improve the therapeutic efficacy of brain glioma in animals. J. Control. Release., 2010 141, 183–192. https://doi.org/10.1016/j.jconrel.2009.09.020 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-5308408","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":368926057,"identity":"967552f4-5884-460f-a57b-397bb2812fee","order_by":0,"name":"Tamilselvan 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1","display":"","copyAsset":false,"role":"figure","size":455842,"visible":true,"origin":"","legend":"\u003cp\u003e(A) AFM 2D image of GAL-CS-NPs, (B) 3D image of GAL-CS-NPs, (C) TEM image of GAL-CS-NPs, (E) Selected area electron diffraction pattern of GAL-CS-NPs.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5308408/v1/bcc2c262fc82566126a9e949.png"},{"id":68171535,"identity":"c4dfbfc2-cfe9-4e1d-aeb0-266bf30577be","added_by":"auto","created_at":"2024-11-04 10:35:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":49298,"visible":true,"origin":"","legend":"\u003cp\u003eDSC thermograms of (A) CS, (B) GAL, (C) Physical mixture (Drug and polymer), (D) GAL-CS-NPs.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5308408/v1/1390e96f640e3acc9ef2fbcf.png"},{"id":68171406,"identity":"f00a7f42-40ca-4d26-8f91-907f2f81d435","added_by":"auto","created_at":"2024-11-04 10:27:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":226555,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of SH-SY5Y cells, (A) incubated for 12 hrs, (B) incubated for 24 hrs.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5308408/v1/8676c40e7d19d25a7a6d610d.png"},{"id":68171534,"identity":"1ce66cd7-969a-49cd-8a2a-726455b46941","added_by":"auto","created_at":"2024-11-04 10:35:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":162616,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability assay, (A) SH-SY5Y cells incubated for 12 hr, (B) 24 hr with GAL-Solution, (C) GAL-CS-NPs, (D) PS-80-GAL-CS-NPs at 10 and 100µg/mL concentration.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5308408/v1/676af1b9dcdfbcb0bb5f456f.png"},{"id":68171409,"identity":"4e3f0aca-f1ce-481f-9c5c-56edf21fb9f1","added_by":"auto","created_at":"2024-11-04 10:27:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":186699,"visible":true,"origin":"","legend":"\u003cp\u003e(A) In-vitro release of GAL-Solution and GAL-CS-NPs, (B) Plasma concentrations of GAL-Solution, GAL-NPs-PS-80 and GAL-NPs after oral administration at different time intervals, (C) GAL concentration (ng/g tissue) in brain at 1st, 4th and 8th hr after oral administration, (D) Brain to plasma ratio of GAL-Solution, GAL-NPs and GAL-NPs-PS-80 formulations at different time intervals after oral administration.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5308408/v1/3691ca003fbf4f1c06af204b.png"},{"id":68171407,"identity":"c21edbde-3cda-4590-b180-bfc837c9cc8e","added_by":"auto","created_at":"2024-11-04 10:27:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":604562,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic images of cerebral sections of rat brain (10x), (A) control rat brain, (B) rat exposed to GAL-Solution (1mg/kg), (C) uncoated GAL-CS-NPs, (D) PS-80 coated GAL-CS-NPs\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5308408/v1/58a6f802292ad00c6f9129a1.png"},{"id":68664866,"identity":"7671b7bb-82c3-4a3d-83a1-a5d5f285f193","added_by":"auto","created_at":"2024-11-10 18:16:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2461113,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5308408/v1/a221a8e4-8a58-4166-8e70-591a34437511.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Polysorbate-80 coated Galantamine-Chitosan nanoparticles for enhanced safety and efficacy: Pharmacokinetics and Brain distribution in Rats","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAD disease is a neurologically degenerative disorder becoming an increasingly difficult challenge to families, the healthcare industry, and caregivers [1]. Even though a variety of marketed drugs are authorized for AD, it remains a significant area of unmet medical need due to limitations like higher doses required for therapeutic effect, limited bioavailability, and poor absorption in the brain [2]. Also, it has been reported as it has significant peripheral side effects owing to increased2 absorption by normal cells, and it has difficulty in penetration at blood brain barrier (BBB) [3].\u003c/p\u003e \u003cp\u003eGalantamine (GAL) is a competitive acetylcholinesterase inhibitor used for a broad spectrum of AD symptoms and authorized by the USA FDA (\u0026ldquo;Food and Drug Administration\u0026rdquo;) and the EMA (\u0026ldquo;European Medicines Agency\u0026rdquo;) [4]. Dual method of action on the cholinergic system, inhibiting acetylcholinesterase and modulating nicotinic acetylcholine receptors allosterically [5]. In recent research, it was shown that GAL could also inhibit Aβ aggregation as well as Aβ induction of neuronal apoptosis [6,7]. But the drug\u0026rsquo;s clinical utility is hindered by its poor retention in the CNS (central nervous system) and the complexity of transporting it around BBB [8,9]. Several therapeutic strategies have been employed to enhance CNS bioavailability [10]. RMT (\u0026ldquo;Receptor-Mediated Transcytosis\u0026rdquo;) or AMT (\u0026ldquo;Absorptive-Mediated Transcytosis\u0026rdquo;) through the cerebral endothelium is the outcome of medication combined with a particular brain transport targeted [11]. Biodegradable polymeric drug carriers like nanoparticles (NPs) and liposome composed of natural, synthetic, semi-synthetic polymers and lipids have been applied to the target brain [12]. Among these, polymeric NPs are promising carrier since it has the capacity to open the tight junctions of BBB. Also, it could prolong the drug release and protect the drug from enzymatic degradation [13]. It has been reported that NPs containing hydrophilic polymers have the additional advantage of longer blood circulation, which would enhance extravasation \u0026amp; passive targeting [14].\u003c/p\u003e \u003cp\u003eNumerous materials, including natural and synthetic polymers, are used as drug carriers; among them, the natural polysaccharide chitosan (CS) has gained popularity due to its outstanding set of physical and biological properties [15]. CS is a natural polysaccharide composed of copolymers of glucosamine and N-acetylglycosamine. Recently CS has arisen as a promising alternative for improving the transport of such macromolecules across biological barriers for drug delivery and biomedical applications [16]. It was reported that the overcoating of the nanoparticles with polysorbates-80 (PS-80) capable of transporting drug across BBB the precise mechanism is not well established [17]. The overcoating of the particles lead to adsorption of apolipoprotein E from blood plasma foiiowed by receptor mediated endocytosis in then present investigation, we attempted to asses polysorbate \u0026minus;\u0026thinsp;80 coated crosslinked with chitosan nanoparticles for brain targeting of GAL. The in-vivo pharmacokinetics and brain distribution analyses were performed in rats to calculate the efficacy of PS-80-coated GAL-CS-NPs in the brain\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Preparation and optimization of NPs\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA straightforward, scaled-up version of the previously reported ionotropic gelation process was used to create CS-NPs [20]. In order to create NPs, cationic polyelectrolyte polymer CS is utilized. The NPs were created by regulating the gelation\u0026rsquo;s interaction with the polyanion TPP, which decreased the solubility of CS in water. The NPs were created by regulating the gelation\u0026rsquo;s interaction with the polyanion TPP, which decreased the solubility of CS in water. The rate-limiting step, which regulates the size and size distribution of NPs, is the CS/TPP ratio [21]. In order to obtain NPs under 200 nm, various ratios of CS/TPP were used in the formulation of NPs. To attain the ideal CS and TPP ratio, several concentrations of GAL-loaded CS and TPP NPs were produced via the ionic interaction of positively charged CS and negatively charged TPP.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Physiochemical characterization of NPs\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe zeta potential, PDI, mean particle size, and drug loading of uncoated GAL-CS-NPs were studied (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The mean particle size and PDI varied from 102\u0026thinsp;\u0026plusmn;\u0026thinsp;4 to 4852\u0026thinsp;\u0026plusmn;\u0026thinsp;8 nm and 0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 to 0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31, respectively, depending upon the concentration of TPP and CS. The zeta potential was in the range of 31.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 to 40.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 mV, and the drug loading varied from 42.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 to 70.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5%. Later PS-80 was used to modify the surface of GAL-CS-NPs. Considering the fact that polymeric NPs modified by PS-80 were taken up rapidly by brain micro vessel endothelial cells, an attempt was made to enhance the uptake of GAL in the brain by modifying the surface of NPs using PS-80. The mean particle size of PS-80 coated GAL-CS-NPs containing 0.6 and 0.2% of CS was found to be 98\u0026thinsp;\u0026plusmn;\u0026thinsp;4 and 126\u0026thinsp;\u0026plusmn;\u0026thinsp;3 nm, respectively. Zeta potentials of 31.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 and 40.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 mV was discovered. After the NPs were coated with PS-80, there was a modest increase in particle size and a decrease in zeta potential owing to obscuring the NPs\u0026rsquo; surface from the additional layer of PS-80 coating (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). AFM and TEM were also used to characterize the GAL-CS-NPs. The acquired data indicates that individual particle sizes were in the 20\u0026ndash;25 nm range. Because CS-NP has a high swelling capacity, the diameter of the particles assessed using the dynamic light scattering (DLS) approach was larger than the size calculated using TEM and AFM. The Zetasizer evaluates the hydrodynamic diameter based on how a particle diffuses inside a fluid. The particle\u0026rsquo;s surface structure, as well as the particle\u0026rsquo;s size, determine the hydrodynamic diameter. The GAL-CS-NPs 3 formulation with the small particle size and zeta potential value of greater than +\u0026thinsp;30 mV, which indicates the stability of NPs was selected as the optimized formulation. The external morphology of the optimized uncoated NPs was studied using TEM and AFM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The TEM pictures demonstrated that the NPs were spherical in form without any aggregates of particles with a size range of 20 nm scale (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), and it was further supported by the AFM image (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u0026amp;D), which revealed particles with a smooth surface devoid of any apparent pinholes or fractures. SAED of drug-loaded NPs in TEM revealed the amorphous state of GAL in CS-NPs. The SAED image clearly demonstrated the absence of a ring pattern owing to the presence of GAL in amorphous from (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This investigation provides proof that the CS-NPs\u0026rsquo; integrated GAL was an amorphous substance.\u003c/p\u003e \u003c/div\u003e \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\u003eComposition of CS-NPs, particle size, PDI, zeta potential and drug loading.\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=\"char\" char=\"\u0026plusmn;\" 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\"\u003e \u003cp\u003eFormulations\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCS (1%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTPP (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSize (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePDI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eZeta Potential\u003c/p\u003e \u003cp\u003e(mV)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEntrapment efficiency (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAL-CS-NPs 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2628\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e46.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAL-CS-NPs 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2444\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e12.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e55.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAL-CS-NPs 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e98\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e40.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e65.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAL-CS-NPs 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e126\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e31.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e70.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAL-CS-NPs 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2574\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e52.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAL-CS-NPs 6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2953\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e4.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e42.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAL-CS-NPs 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e4731\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e47.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAL-CS-NPs 8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2852\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e55.06\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAL-CS-NPs 9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2764\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e43.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\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\u003eParticle size and zeta potential of GAL-CS-NPs after PS-80 coating.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFormulation code\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSize (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePDI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eZeta Potential (mv)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePS-80-GAL-CS-NPs 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e115\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e31.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePS-80-GAL-CS-NPs 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e126\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e26\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe crystalline character of the NPs was investigated using DSC and SAED investigations. The thermograms for CS, GAL, physical mixture, and GAL-CS-NPs were developed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The broad endothermic peak of pure GAL at 271.77 \u0026ordm;C was absent in the thermogram of GAL-CS-NPs, indicating the absence of crystalline material in the NPs. The absence of a peak might be due to the inhibition of crystallization of GAL by polymer during NPs preparation. This leads to the conclusion that the medicine included in the NPs was in an amorphous, molecular dispersion, solid solution, or disordered crystalline inside the matrix of the polymer.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. In-vitro drug release study\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn-vitro release behavior of the uncoated GAL-loaded CS-NPs was examined in phosphate buffer (pH 6.5). The release profile demonstrated the cumulative drug release for the optimized formulations GAL-CS-NPs 3 and GAL-CS-NPs 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The release of drug from drug-loaded NPs varied from 56.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 to 65.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7% over a period of 24 hrs. It was apparent from the release profile that the NPs showed an initial burst release around 17.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 and 16.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8% at the end of 1st hr and followed by a slow-release pattern of the drug was found to be 56.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 and 65.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7%. The initial burst of GAL release may have been caused by adsorbed GAL on the NP surface, and the entrapped GAL slowly released from the NPs after that. The release data were fitted to the Korsmeyer-Peppas model, as shown by higher regression coefficients (r2-values), which were determined to be 0.8876 and 0.8975 for GAL-CS-NPs 3 and GAL-CS-NPs 4, respectively, in order to study the manner of drug release from CS-NPs. The obtained \u0026lsquo;n\u0026rsquo; values 0.290 and 0.324 suggested that the drug releases followed the Fickian diffusion mechanism.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Storage stability studies\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe appearance of PS-80-GAL-CS-NPs was found to be pale white powder during the lyophilization process, and, there were no changes in appearance even after 6th month of storage at room temperature. The particle size of PS-80-GAL-CS-NPs was found to be 116\u0026thinsp;\u0026plusmn;\u0026thinsp;4 on the initial day of preparation. After storage for 6 months, the size of particles was slightly increased in size as 125\u0026thinsp;\u0026plusmn;\u0026thinsp;5, 138\u0026thinsp;\u0026plusmn;\u0026thinsp;4, 149\u0026thinsp;\u0026plusmn;\u0026thinsp;9 and 154\u0026thinsp;\u0026plusmn;\u0026thinsp;6 nm in 1st, 2nd, 4th, and 6th months, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The drug entrapment efficiency also was found to be stable at 64.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6, 65.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2, 64.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 and 66.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 at 1st, 2nd, 4th and 6th months, respectively.\u003c/p\u003e \u003c/div\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\u003eStability studies of PS-80 coated GAL loaded CS-NPs.\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=\"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=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTemperature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEvaluation\u003c/p\u003e \u003cp\u003eparameter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003eObservation (months)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003e24\u003c/b\u003e\u003csup\u003e\u003cb\u003eo\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eC\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(RH\u0026thinsp;=\u0026thinsp;65%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhysical appearance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePale white powder\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo change\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo change\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo change\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo change\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParticle size (nm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e116\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e125\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e138\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e149\u0026thinsp;\u0026plusmn;\u0026thinsp;9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e154\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEntrapment efficiency (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e65.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e66.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Stability of drug in SGF\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe stability of GAL in SGF was examined. It was found that the percentage of drug remaining was found to be 72\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 and 75.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1%. The obtained result shows that the drug was protected from gastric enzymatic degradation for up to 3 hrs. It could be because PS-80 has lengthy chains with dense surfaces that act as a protective brush to stop the medicine from degrading.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. In-vitro cell study\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1. MTT Assay\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCytotoxicity of uncoated GAL-NPs and PS-80 coated GAL-NPs was evaluated for screening novel compounds for neurotoxicity properties. For 12 and 24 h, the cells were treated with drug solution, uncoated CS-NPs, \u0026amp; CS-NPs coated with PS-80 at concentrations of 10 and 100 g/mL. The data showed that neither uncoated nor PS-80 coated GAL loaded NPs were found to be non-toxic on these SH-SY-5Y cells, even at the higher concentration of the formulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The cell viability was above 90% for all assays.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2. Cellular uptake study\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFluorescence microscopy was used to examine the capacity of NPs-Rho loaded CS-NPs to be internally incorporated into SH-SY5Y human neuroblastoma cells [23]. The cellular uptake of uncoated Rho-CS-NPs was studied at the 1st and 4th hr. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026amp;B). At 1st hr., the uptake of Rho on the cells was not visible, and after a few hrs., the uptake of Rho was seen in the surrounding of the cell membrane. With regard to Rho-NP coated with PS-80, the internalization of Rho was clearly seen inside the membranes after 1st and 4th hr. incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The internalization of Rho was also seen in the entire membrane\u0026rsquo;s cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The internalization of Rho-PS-80-CS-NPs was higher with time when compared to the uncoated and free Rho. Positively charged CS and negatively charged cell membranes had strong ionic contact, which led to increased internalization. Further, the other possibility of internalization happened by the presence of PS-80 on the surface of NPs which got adsorbed on the apolipoproteins present on the cell surface through receptor-mediated endocytosis. Hence, NPs may be an effective and safe delivery systems for the oral administration of GAL.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.7. In-vivo studies\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1. Pharmacokinetic studies\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWister male rats were used for in-vivo pharmacokinetics and tissue distribution experiments. Following oral administration of GAL solution, GAL-NPs, and GNP-PS-80, the plasma and tissue concentration of GAL at various time points was measured. With the aid of WinNonlin, the pharmacokinetic parameters were computed using non-compartmental analysis (version 5.1). The pharmacokinetic parameters such as Cmax, tmax, AUC0- 24, t1/2, and MRT (hr) were analyzed. Different formulations\u0026rsquo; plasma concentration vs. time profiles were shown. Rats who were given GAL-NPs had a greater plasma concentration of GAL than rats that were given free GAL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The Cmax of GAL after administration with GAL-NPs was significantly higher than the Cmax obtained after administration of GAL solution (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and a 5.03-fold increase was observed. A delayed Tmax reveals a prolonged release pattern of GAL-CS-NPs, and a rise in Cmax following treatment via NPs indicates an increase in the quantity of GAL that has entered the systemic circulation. This shows that GAL-NPs improve absorption by lowering the first-pass metabolism, resulting in enhanced medication availability for extended periods of time. GAL solution and GAL-NPs were found to have AUCs of 1064.12 and 6877.7 ng/mL, respectively. When given as NPs at 24 h, more GAL was seen in the plasma than in the GAL solution, indicating a sustained release pattern for CS-NPs. When compared to the plasma concentration profile of GAL-NPs, the plasma drug concentration profile obtained after administering PS-80 coated NPs showed a lower amount of GAL in blood, indicating that the PS-80 coating protected the release of drug in the plasma, enhancing the presence of GAL in the brain.[26]\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2. Brain distribution studies\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe concentration of GAL in the brain was estimated by HPLC. The concentration of GAL (ng/tissue) was achieved in the brain at different time points after oral administration of GAL solution, GAL-NPs and GAL-NP-PS-80 at 1, 4 and 8th hrs post-administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The representing chromatogram of GAL concentration from GNP-PS-80 in the brain at different time points 1, 4 and 8 hrs are recorded (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The NPs formulation facilitated the improved uptake of the drug and availability in both plasma and brain tissue. The drug concentration was found to be 32.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2, 28.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 and 17.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5 ng/tissue after 1, 4 and 8th hrs. The plain GAL solution showed a decrease in the availability of the drug. When administered as NPs, the drug concentration showed a little increase in the brain, indicating the sustained release of CS-NPs. The concentration of NPs was found to be 226.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3, 114.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3, and 110.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9 ng/tissue in the case of PS-80 coated NPs, showing enhanced drug availability in the brain at all time points. PS-80 coating indicated the enhanced uptake of GAL in the brain through receptor-mediated endocytosis and showed the importance of surface properties in the biodistribution of NPs. Additionally, adsorptive-mediated transcytosis induced by ionic interaction between the negatively charged membrane surface and the positive charge of NPs facilitated an increased permeation across BBB.[22]\u003c/p\u003e \u003cp\u003eThe ratio of drug concentration in the brain to that in plasma is often used as a targeting indicator. Rats were used to study the ratio of brain to plasma concentration of the GAL formulations (GAL-solution, GAL-NPs, and GNP-PS-80) at various time points after oral treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The findings show that the GNP-PS-80 formulation\u0026rsquo;s brain-to-plasma ratio was consistently larger than one at all time periods (1, 4 and 8th hrs.). The preferential localization of GAL in the brain for CS-NPs coated with PS-80 when compared with the GAL-solution and uncoated GAL-NPs can be attributed to endocytosis. These results indicate that the PS-80 coated CS-NPs after oral administration enhanced the delivery of GAL to the brain.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.7.3. Histopathological studies\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAfter administering NPs orally to the cerebral area, photomicrographs of brain slices were examined for morphological alterations, including inflammation of neurons. The GAL-Solution treated GAL-NPs and PS-80 coated GAL-NPs did not exhibit any morphological abnormalities, such as vacuolization/spongiosis and neuronal degeneration in the morphological sections, which mimic brain sections of normal nerve cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Therefore, it is evident that the polymeric NPs with PS-80 coated were safe for the brain [24]\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Materials and Methods","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Materials\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eChitosan low molecular weight was purchased from Sigma-Aldrich, USA, sodium tripolyphosphate (TPP) was purchased from Loba Cheme, India. The Glacial acetic acid was obtained from Fisher Scientific, USA. Galantamine hydrobromide was obtained as a gift sample by Alembic, India. Dialysis membrane with a molecular weight cut off 12,000\u0026ndash;14,000 Daltons was purchased from HI Media Laboratories, India.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Synthesis of nanoparticles\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe ionic gelation process was used to create GLA-CS-NPs [18]. Determinate weights of CS (0.2, 0.4 and 0.6%) were dissolved in glacial acetic acid 1 percent (v/v). Aqueous sodium tri poly phosphate (TPP) solution was added in a dropwise manner at concentrations of 0.2, 0.4, and 0.6 percent after GAL was added to CS while being constantly magnetically stirred. For 30 min, the mixture was continuously stirred with magnets. To eliminate excessive levels of TPP and unencapsulated free GAL, the suspension of NPs was centrifuged at 13,000rpm for 30 min at 4◦C using an Ultracentrifuge. The pellets have been dispersed within deionized water. Finally, NPs have been lyophilized with a freeze dryer for 24 hrs. (Lyodel, India) in preparation for powder storage. To prepare fluorescently loaded CS-NPs, Rhodamine-6G was dissolved in ethanol (20 mg/mL) and further combined in 10 mL of CS solution (0.6%) and cross-linking with 0.2percent w/v TPP solution at room temperature with constant stirring for 30 min at 1,000 rpm. The NPs have been centrifuged at 13,000rpm at 4\u0026ordm;C for 30min, and the pellet was separated and stored until further study.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Coating of GLA-CS-NPs\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe NPs coating was conducted as per the procedure defined by [19]. The freeze-dried drug-loaded NPs (20 mg per ml) were resuspended in PBS (\u0026ldquo;Phosphate Buffered Saline\u0026rdquo;) under constant stirring at 50 rpm (Remi Motors, India). Then PS-80 was added to have a final solution of 1 percent (v/v) mixture and was incubated for 30min. The freeze-dried NPs were subjected to zeta potential, particle size, along with drug loading characterizations.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Physiochemical characterization of GAL-CS-NPs\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1. Zeta potential and particle size measurement\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe average hydrodynamic diameter as well as PDI (\u0026ldquo;Polydispersity Index\u0026rdquo;) of the formulated NPs were examined by Zetasizer Nano ZS90 (Malvern Instruments Ltd., United Kingdoms), using DLS (\u0026ldquo;Dynamic Light Scattering\u0026rdquo;) technique. For the purpose of measuring particle size, a 1ml sample of NPs dispersion was deposited in disposable cuvettes. Each experiment was carried out three times. The Malvern Zetasizer (Nano ZS90, Malvern Instruments) was used to evaluate electrophoretic mobility (zeta potential) at a temperature of 25\u0026ordm;C. Double-distilled water was used to dilute the samples.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.4.2. TEM (Transmission Electron Microscopic) analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe prepared NPs\u0026rsquo; morphology was assessed using a TEM fitted with a SAED (Selected Area Electron Diffraction) Pattern. An NPs suspension drop was placed on a copper grid, and the scanning was conducted at 80kv (\u0026ldquo;JOEL JEM 2100, Japan\u0026rdquo;). The morphology of the copper grid was examined while it was in the vacuum chamber of a TEM with the grid-connected to a sample holder.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.4.3. Atomic force microscopic (AFM) determination\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eUnder typical air circumstances, an AFM (\u0026ldquo;Nova NTEGRA prima, Russia\u0026rdquo;) was used to observe the surface characteristics of drug-loaded NPs. The scan rate was set at 2 Hz. The samples were placed on glass slides after being diluted ten times with distilled water and vacuum dried at 25\u0026deg;C for 24 hrs. AFM image analysis software (\u0026ldquo;Multimode scanning probe microscope\u0026rdquo;) was used to estimate height.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.4.4. Drug encapsulation determination\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe drug EE (Encapsulation Efficiency) of Gal-CS-NPs was examined as per the approach depicted (Vila et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Each batch included 50 mg of drug-loaded CS-NPs, which were digested for 24 h in a 20 mL solution of 0.1 N HCl and 1:1 v/v ethanol. The particles have been separated using centrifugation at 13,000rpm, and the drug concentration in the supernatant was examined using Ultraviolet spectrophotometry at 289nm.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\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 \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e%\u003c/b\u003e EE=(Weight of total drug-Weight of free drug)/(Weight of total drug)\u0026times;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.4.5. Differential scanning calorimetric (DSC) analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eGAL\u0026rsquo;s physical state inside the NPs was described by DSC. The thermogram of pure GAL, CS, physical mixture and GAL-CS-NPs was conducted using DSC. A standard aluminum pan was utilized to contain the sample (5mg), which was then crimped as well as heated at a rate of 10\u0026ordm;C/in from 4 to 450\u0026ordm;C while nitrogen was being purged (20 mL/ min). The empty sealed aluminum pan was employed as a reference.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.5. In-vitro drug release\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA revised dialysis approach was applied to assess the in-vitro release of GAL-CS-NPs (Hu et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). A dialysis bag was filled with the NP\u0026rsquo;s suspension, which contained 2 mL or 2 mg of GAL. Both the ends of the dialysis bag were tied using membrane clips and placed into 20mL of \u0026ldquo;phosphate buffer\u0026rdquo; (0.1 M, pH 6.5), kept at 37\u0026ordm;C with continuous magnetic stirring. Aliquots from the releasing medium were taken out at predetermined times and replaced with the equivalent volume of phosphate buffer. GAL at 289 nm was measured spectrophotometrically in the sample.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.6. In-vitro release kinetics\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDDSolver was used to calculate the drug release data, and the resulting data were fitted to the Korsmeyer-Peppas exponential equation to determine the mechanism of drug release, in which Q indicates the proportion of the drug released at time t and k denotes a constant incorporating the structural and geometric properties of the device under examination (Zhang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The mode of drug transport from the dosage form is significantly indicated by the diffusional exponent \u0026#119899;.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Storage stability studies\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe storage stability of PS-80-GAL-CS-NPs was examined according to ICH (\u0026ldquo;International Conference on Harmonization\u0026rdquo;) recommendations. The formulations were stored at room temperature (24oC, RH\u0026thinsp;=\u0026thinsp;65%) in the dark for six months. The physical appearance, average particle size, and entrapment efficacy of the stored formulations were examined during the 1st, 2nd, 4th and 6th months to determine their stability.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Stability of drug in SGF (Stimulated Gastric Fluid)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe GAL stability was assessed in SGF, prepared as per \u0026ldquo;British Pharmacopoeia\u0026rdquo; (BP). In a nutshell, 80mL of 1M HCl was used to dissolve 2g of NaCl and 3.2g of pepsin before the volume was increased to 1000mL and the pH was set to 1.2. Typically, 15 mL of SGF was used to suspend 50 mg of every formulation, and the mixture was then put into screw-capped tubes. The water shaker bath was maintained at 37\u0026deg;C with the tubes. The suspensions have been centrifuged at 13,000 rpm for 40 min after 3 h of incubation in SGF, and the supernatants were discarded. The sediment was disseminated in the solvent, and the drug concentration was measured using UV light at 289 nm.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.9. In-vitro cell studies\u003c/h2\u003e \u003cdiv id=\"Sec30\" class=\"Section3\"\u003e \u003ch2\u003e3.9.1. MTT assay\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eNational Center for Cell Science (NCCS), Pune, provided the human SH-SY5Y cells. Cells were cultured in MEM with non-essential amino acids, ham F-12, 10 percent fetal bovine serum, 2 mmol/L L-glutamine, penicillin (100 U/mL), and streptomycin (100\u0026micro;g/mL ) in a humid atmosphere at 37\u0026deg;C and 5 percent CO2. Every week, the medium was changed twice. Cell viability was evaluated with the \u0026ldquo;3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide\u0026rdquo; (MTT) test. SH-SY5Y cells were planted at a density of 1\u0026times;104 cells per well in 96-well plates for 24 h under a 10 percent serum condition. Cells were kept in serum-free media for 16 h after the serum concentration was gradually reduced (5%) over a period of days. A normal control group, a placebo, and three different formulations of galantamine at doses of 10 \u0026amp;100 \u0026micro;g each were divided among the wells in triplicate. Cells were exposed to drug formulations for 24 h. MTT was produced at a concentration of 5 mg/mL, and 20\u0026micro;L of the MTT solution was applied to each well before being incubated for four hours. After the MTT had been incubated, the medium containing the MTT was taken out, and each well-received 50\u0026micro;L of DMSO was to dissolve the formazan crystals. At 570 nm, optical density was investigated. In comparison to the control, percentage toxicity was calculated.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section3\"\u003e \u003ch2\u003e3.9.2. Cellular uptake study\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eConfluent SH-SY-5Y was used in the qualitative investigation. Human neuroblastoma cells were grown in MEM with non-essential amino acids, ham F-12, 10% fetal bovine serum, 2 mmol/L L-glutamine, penicillin (100 U/mL), and streptomycin (100 \u0026micro;g/mL) and kept in a humid atmosphere at 37\u0026deg;C and 5% CO2. Every week, the medium was changed twice. SH-SY-5Y cells were seeded in 96-well plates at a density of 1\u0026times;104 cells per well, and they were subsequently incubated at 37 C with rhodamine-6 (Rho) loaded NPs. The cells were examined using fluorescence microscopy after being rinsed 3 times with cold PBS.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e3.10. Bio-analytical method development\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe concentration of GAL was quantified using HPLC (\u0026ldquo;High-Performance Liquid Chromatography\u0026rdquo;) (waters 2489) equipped with a UV detector at 289 nm with a reversed-phase column (Gemini C18, 5\u0026micro;,50x30mm Phenomenex). The mobile phase consists of 0.01M ammonium acetate buffer (pH 3) and ACN (94:6 v/v) at the flow rate of 0.5ml/minute. The extraction of GAL from tissue was conducted using liquid\u0026ndash;a liquid extraction technique. Rat tissue homogenate (100\u0026micro;L) and 20\u0026micro;L of a solution containing 1 M sodium hydroxide were mixed and vortexed for 60 s. This mixture received 0.7 mL of tetra butyl methyl ether, which was then centrifuged at 10,000 rpm for 5 min. Following separation, the organic layer was exposed to a nitrogen gas stream while being enclosed by a turbo vap LV (Biotage USA) at 40\u0026deg;C. Finally, 20\u0026micro;L of the reconstituted mobile phase was used for HPLC analysis after the dried residue was re-formed with 100\u0026micro;L of the mobile phase.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section2\"\u003e \u003ch2\u003e3.11. In-vivo evaluations\u003c/h2\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section2\"\u003e \u003ch2\u003e3.11.1. Pharmacokinetic study\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe in-vivo pharmacokinetic tests were conducted on male Wistar rats weighing 200\u0026ndash;250g. PSG Institutional Animal Ethics Committee (Proposal No. 197/2013/IAEC), located in Coimbatore, examined, and approved the protocol for the animal trials. The animals were housed in a well-ventilated animal home with a sterile paddy husk cage, a regular diurnal cycle, and full access to food and water. The night before the experiment, the animals were given permission to fast. Three groups of six animals each were formed from the total number of animals. The animals were given a dosage of 1 mg/kg orally using a gastric lavage needle. GAL solution was given to group 1 suspended in PBS 7.4, GAL-NPs were given to group 2 uncoated, and PS-80 coated GAL-NPs were given to group 3. At 60, 120, 240, 480, 720, and 1440 min after treatment, retro-orbital venous plexus punctures were used to collect the blood samples (500 L) into microcentrifuge tubes containing sodium citrate. The blood samples were centrifuged at 4,000 rpm for ten mins. For further studies, the separated plasma was stored at a temperature of \u0026minus;\u0026thinsp;20\u0026deg;C\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec35\" class=\"Section2\"\u003e \u003ch2\u003e3.11.2. Brain distribution study\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe Wister rats used in the tissue distribution assays weighed between 200 and 250 g. Three groups of six rats each were created from the group of rats. The drug was administered through the oral route. Groups 2 and 3 each got uncoated GAL-NPs at a dosage of 1 mg/kg, whereas Group 1 received a GAL solution. Three animals from each group were sacrificed after the first, fourth, and eighth hours of treatment. The brain\u0026rsquo;s target tissue was then removed and homogenized with ice-cold PBS 7.4 in a tissue homogenizer (Remi) (Gaur et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). HPLC, in conjunction with a UV detector, was applied to assessed the content of drug in the organs.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec36\" class=\"Section2\"\u003e \u003ch2\u003e3.11.3. Histopathological analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eOn Wister rats, a histopathological study was done. After 24 h, animals were slaughtered, and the brains were taken out. The brain tissue was embedded in paraffin after being treated in a 10% formaldehyde solution. 7 mm thick paraffin slices were created for microscopy research. Finally, after being submerged in water, the tissues were stained with hematoxylin and eosin for ten minutes at 60\u0026deg;C. The discolored areas were improved for dehydration using various grades of alcohol after being rinsed in running water to eliminate the surplus stain. Finally, the slides were made permanent by clearing them with xylene and mounting them with DPX. The slices were examined for the presence of neuronal degeneration, lipifuscin deposits, and vacuolization/spongiosis\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBy using a modified ionic gelation technique and optimizing the particle size\u0026thinsp;\u0026gt;\u0026thinsp;200 nm, PS-80 coated GAL-loaded CS-NPs were created, increasing their absorption in the brain. The pharmacokinetic data indicate a sustained release profile of GAL from CS-NPs when compared to GAL-Solution. Distribution studies to the brain proved that PS-80 coated GAL-NPs enhanced the drug availability in the brain for up to 8 hrs when compared with the uncoated NPs after oral administration. Therefore, the polymeric NPs developed after surface coating with PS-80 represent a promising approach to overcoming the BBB issues for the brain delivery of drugs to treat other CNS disorders.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: T.N, and V.R.C;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData collection and curing: T.N A.K, H.R, P.K.G and S.K;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrimary Draft: T.N,V.R.C, K.A and K.S;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSoftware: T.N, H.R, K.S and A.K;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinal Draft: T.N, V.R.C, H.R, P.K.G, K.S, A.K\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The authors are also thankful to Indian Council of Medical Research (ICMR), New Delhi for the research grant titled “Development of nanoparticleculate drug delivery systems to treat Alzheimer’s disease” Ref No: 35/36/2010/Nano-BMS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003ePSG Institutional Animal Ethics Committee (Proposal No. 197/2013/IAEC), located in Coimbatore, examined, and approved the protocol for the animal trials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e The authors are thankful to PSG College of Pharamcy, Coimbatore for providing facilities to carry out the work\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKumar, A.; Sidhu, J.; Goyal, A. Alzheimer Disease. In StatPearls; StatPearls Publishing: Treasure Island, FL, USA, 2020. https://www.ncbi.nlm.nih.gov/books/NBK499922\u003c/li\u003e\n\u003cli\u003eDong, X., Current Strategies for Brain Drug Delivery. Theranostics.2018.8,1481\u0026ndash;1493. https://doi.org/10.7150/thno.21254\u003c/li\u003e\n\u003cli\u003eZhao, D.; Yu, S.; Sun, B,; Gao, S, ; Guo, S, ; Zhao, K., 2018. Biomedical Applications of Chitosan and Its Derivative Nanoparticles. Polymers (Basel). 10, 462. https://doi.org/10.3390/polym10040462\u003c/li\u003e\n\u003cli\u003eRubin, L.L,; Staddon, J.M,;. The cell biology of the blood-brain barrier. Annu. Rev. Neurosci. 1999, 22, 11\u0026ndash;28. https://doi.org/10.1146/annurev.neuro.\u003c/li\u003e\n\u003cli\u003eLilienfeld, S. Galantamine - a Novel Cholinergic Drug with a Unique Dual Mode of Action for the Treatment of Patients with Alzheimer\u0026rsquo;s Disease. CNS Drug Rev. 2006, 8, 159\u0026ndash;176. https://doi.org/10.1111/j.1527-3458.2002.tb00221.x\u003c/li\u003e\n\u003cli\u003eMatharu, B,; Gibson, G,; Parsons, R,; Huckerby, T.N,; Moore, S.A,; Cooper, L.J,; Millichamp, R,; Allsop, D,; Austen, B., Galantamine inhibits \u0026beta;-amyloid aggregation and cytotoxicity. J. Neurol. Sci, 2009. 280, 49\u0026ndash;58. https://doi.org/10.1016/j.jns.2009.01.024\u003c/li\u003e\n\u003cli\u003eDe Boer, A.G,; Gaillard, P.J,; Drug Targeting to the Brain. Annu. Rev. Pharmacol. Toxicol,2007. 47, 323\u0026ndash;355. https://doi.org/10.1146/annurev.pharmtox.47.120505.105237\u003c/li\u003e\n\u003cli\u003eLiu, X,; Xu, K,; Yan, M,; Wang, Y,; Zheng, X, Protective effects of galantamine against A\u0026beta;-induced PC12 cell apoptosis by preventing mitochondrial dysfunction and endoplasmic reticulum stress. Neurochem. Int, 2010. 57, 588\u0026ndash;599. https://doi.org/10.1016/j.neuint.2010.07.007\u003c/li\u003e\n\u003cli\u003eRoney, C,; Kulkarni, P,; Arora, V,; Antich, P,; Bonte, F., Wu, A., Mallikarjuana, N.N., Manohar, S., Liang, H.-F., Kulkarni, A.R., Sung, H.-W., Sairam, M., Aminabhavi, T.M., Targeted nanoparticles for drug delivery through the blood\u0026ndash;brain barrier for Alzheimer\u0026rsquo;s disease. J. Control. Release, 2005, 108, 193\u0026ndash;214. https://doi.org/10.1016/j.jconrel.2005.07.024\u003c/li\u003e\n\u003cli\u003eHeinrich, M,; Lee Teoh, H,;. Galanthamine from snowdrop\u0026mdash;the development of a modern drug against Alzheimer\u0026rsquo;s disease from local Caucasian knowledge. J. Ethnopharmacol, 2004. 92, 147\u0026ndash;162. https://doi.org/10.1016/j.jep.2004.02.012\u003c/li\u003e\n\u003cli\u003eBickel, U,; Yoshikawa, T,; Partridge, W.M., Delivery of peptides and proteins through the blood\u0026ndash;brain barrier. Adv. Drug Deliv. Rev. 2001. 46, 247\u0026ndash;279. https://doi.org/10.1016/S0169-409X(00)00139-3\u003c/li\u003e\n\u003cli\u003eArumugam, K,; Subramanian, G,; Mallayasamy, S,; Averineni, R,; Reddy, M,; Udupa, N., A study of rivastigmine liposomes for delivery into the brain through intranasal route. Acta Pharm. 2008.58, 287\u0026ndash;297. https://doi.org/10.2478/v10007-008-0014-3\u003c/li\u003e\n\u003cli\u003eKreuter, J,; Ramge, P,; Petrov, V,; Hamm, S,; Gelperina, S.E,; Engelhardt, B,; Alyautdin, R,; von Briesen,; Begley, D.J.,. Direct evidence that polysorbate-80-coated poly (butyl cyanoacrylate) nanoparticles deliver drugs to the CNS via specific mechanisms requiring prior binding of drug to the nanoparticles. Pharm. Res. 2003, 20, 409\u0026ndash;16. https://doi.org/10.1023/a:1022604120952\u003c/li\u003e\n\u003cli\u003eHans, M.; Lowman, A. Biodegradable nanoparticles for drug delivery and targeting. Curr. Opin. Solid State Mater. 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Pharm, 1997. 152, 237\u0026ndash;246. https://doi.org/10.1016/S0378-5173(97)00096-3\u003c/li\u003e\n\u003cli\u003eArya, G,; Vandana, M,; Acharya, S;, Sahoo, S.K. Enhanced antiproliferative activity of Herceptin (HER2)-conjugated gemcitabine-loaded chitosan nanoparticle in pancreatic cancer therapy. Nanomedicine Nanotechnology, Biol. Med., 2011 7, 859\u0026ndash;870. https://doi.org/10.1016/j.nano.2011.03.009\u003c/li\u003e\n\u003cli\u003eRamge, P,; Unger, R.E,; Oltrogge, J.B,; Zenker, D,; Begley, D,; Kreuter, J,; Von Briesen, H.,. Polysorbate-80 coating enhances uptake of polybutylcyanoacrylate (PBCA)-nanoparticles by human and bovine primary brain capillary endothelial cells. Eur. J. Neurosci. 2000, 12, 1931\u0026ndash;1940. https://doi.org/10.1046/j.1460-9568.2000.00078.x\u003c/li\u003e\n\u003cli\u003eVila, A,; S\u0026aacute;nchez, A,; Tobı́o, M,; Calvo, P,; Alonso, M.J., Design of biodegradable particles for protein delivery. J. Control. 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Release., 2010 141, 183\u0026ndash;192. https://doi.org/10.1016/j.jconrel.2009.09.020\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alzheimer, brain targeting, blood-brain barrier, galantamine, nanoparticle, neuroblastoma, SH-SY5Y cell lines","lastPublishedDoi":"10.21203/rs.3.rs-5308408/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5308408/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn brain drug delivery, the BBB (Blood-Brain Barrier) has been a great barrier for various active pharmaceutical drugs. Hence, developing suitable drug delivery systems are need of the hour. However, the present study aimed to develop a polysorbate-80 (PS-80) coated galantamine (GAL) loaded chitosan nanoparticles (CS-NPs) with particles less than 200 nm to enhance the drug internalization in the brain. GAL-CS-NPs were prepared using the ionic gelation technique and optimized to obtain a particle size of less than 200nm and further coated wirh PS-80 to obtain polysorbate-80 coated galantamine loaded chitosan nanoparticles (PS-80-GAL-CS-NPs) The physiochemical properties of uncoated GAL-CS-NPs, PS-80 coated GAL-CS-NPs and the \u003cem\u003ein-vitro\u003c/em\u003e evaluations, such as cytotoxicity and cellular internalization in SH-SY-5Y human neuroblastoma cell lines, were studied. The particle size of optimized PS-80-GAL-CS-NPs was observed to be 115±4 nm, and zeta potential was found to be 31.2±1.7 mV. While the drug entrapment efficiency was found to be 65.5±1.2 %. The \u003cem\u003ein-vitro\u003c/em\u003e drug release of PS-80-GAL-CS-NPs was found to be 56.75±1.3 %. However, the cytotoxicity studies did not show any toxicity for the drug concentrations of 10 and 100 µg/mL. PS-80-GAL-CS-NPs facilitated time-dependent GAL uptake in SH-SY-5Y cell lines. Studies conducted in vivo in plasma revealed a steady release of GAL from PS-80-GAL-CS-NPs. The distribution PS-80- GAL-CS-NPs in the brain after oral administration at 1\u003csup\u003est\u003c/sup\u003e, 4\u003csup\u003eth\u003c/sup\u003e and 8\u003csup\u003eth \u003c/sup\u003ehr was found to be 1.7, 3.1 and 2.0 folds higher when compared to the uncoated NPs. Further, the histopathological study did not show any morphological changes in the rat brain. This study indicates that PS-80-GAL-CS-NPs might be a promising delivery method for Alzheimer’s Disease (AD)\u003c/p\u003e","manuscriptTitle":"Polysorbate-80 coated Galantamine-Chitosan nanoparticles for enhanced safety and efficacy: Pharmacokinetics and Brain distribution in Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-04 10:27:42","doi":"10.21203/rs.3.rs-5308408/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"26432b6b-95e6-40a6-82c4-4180fd927864","owner":[],"postedDate":"November 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-25T09:53:07+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-04 10:27:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5308408","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5308408","identity":"rs-5308408","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}