RETRACTED ARTICLE: Evaluating the neuroprotective effect of Spirulina platensis–loaded niosomes against Alzheimer’s disease induced in rats

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is characterized by memory loss, changes in behavior, and avoidance of social bonds. Aluminum is one of the main risk factors in the development of AD. Spirulina platensis (SP) is a microalga that improves motor and cognitive skills and prevents cerebral endothelial damage. SP could be delivered in a more controlled and targeted manner using nanoparticles like niosomes. The purpose of this research was to develop a SP-loaded niosome (SPLN) formulation as a drug delivery system to investigate the effectiveness and toxicity of SP as an AD therapy using the AlCl3-induced AD rat model. A niosomal formulation that consists of Tween 60, cholesterol, and dihexadecyl phosphate in a molar ratio of 1:2:0.1 was chosen as an optimum formulation. AD was induced in rats by orally administering AlCl3. Compared with the AlCl3 control, the group treated with the SPLN formulation showed enhancement of the recognition and working memories by increasing the difference score, the discrimination ratio, and the spontaneous alternation behavior. Additionally, it revealed a significant increase in AchE genes, restored the reduced brain neurotransmitters, and improved brain oxidative status. In conclusion, SPLN formulation could be considered an effective AD therapy.


Introduction
Alzheimer's disease (AD) is a progressive neurodegenerative disease that causes amyloid and tangles to accumulate in the brain [1,2].To date, AD has affected over 25 million people all over the globe [3,4].It is characterized by memory loss, changes in mood and behavior, and avoidance of social bonds [5].One of the most significant heavy metals in the development and spread of AD is aluminum (Al), which is found in a variety of commercially created products [6,7].Al has a direct impact on the nervous system's multiple metabolic cascades [7].It upregulates the expression of AD-related pathogenic genes like interleukin-1, interleukin-6, tumor necrosis factor, acetylcholine esterase, and monoamine oxidase [6,7].In the molecular mechanism of AD pathogenesis, Al causes neurotoxicity and amyloid protein oligomerization [8].
Spirulina platensis (SP) is a type of cyanobacteria that thrives in warm, alkaline freshwater lakes [9].Several studies have shown that SP has potential benefits for brain health [9][10][11].It has antioxidant, anti-inflammatory, and neuroprotective properties [9,10,12].It mitigates mental fatigue via preventing endothelial damage to cerebral blood vessels [10,11].Additionally, it improves motor, language, and cognitive skills [13].Drugs could be delivered in a more controlled and specific manner using nanoparticles like liposomes and niosomes [14][15][16].The drug's bioavailability, efficiency, and selectivity are all enhanced by the nanoparticle delivery system [14,15,17].Since phospholipids are easily oxidized and hydrolyzed, niosomes are superior to liposomes [14,15,18].Niosomes are targeted drug delivery systems that use non-ionic surfactants and cholesterol to increase the delivery of water-soluble drugs such as SP [14,15,17,19].Niosomes have been found to improve drug stability, decrease toxicity, increase circulation time, and enhance target-site uptake [14,15,17,20].The purpose of this research was to develop an SP-loaded niosome (SPLN) formulation as a drug delivery system to investigate the effectiveness and toxicity of SP as an AD therapy using the AlCl 3 -induced AD rat model.Different SPLN formulations were prepared and evaluated to choose the optimum formulation for further studies such as the efficacy and toxicity of SPLN in comparison to oral SP.

Materials
Tween 60, cholesterol, dihexadecyl phosphate, chloroform, and methanol were purchased from Agitech Pharmaceutical Company (Cairo, Egypt).Aluminum chloride anhydrous (AlCl 3 ) was imported from an Indian central drug store.Spirulina platensis was obtained as a green colored pure powder from the Amoun Vet.Company (AVC) for pharmaceutical drugs synthesis, Cairo, Egypt.

Preparation of Spirulina-loaded niosome formulation
The thin-film hydration technique was used to prepare Spirulina-loaded niosomes (SPLN) [15].The calculated amounts of Tween 60, cholesterol, and dihexadecyl phosphate (DDP) were dissolved in a solution (10 ml) of chloroform and methanol (3:1), with a molar ratio of 1:2:0.1.Next, the solution was poured into a flask and evaporated at 100 rpm under vacuum using a Stuart rotary evaporator (RE300, UK).SP hydrated the film at 60 °C for 2h after being dissolved in 10 ml of phosphate buffer (PB).The SPLN formulation was ultrasonicated for 30 min using a Sonix (IL, USA).Isolation of SPLN pellets required 1h of centrifugation at 15,000 rpm in a centrifuge (SIGMA, Germany).PB (10 ml) was added to SPLN pellets and maintained at 4 °C.

Morphology of SPLN formulation
The formation and shape of SPLN vesicles were studied with transmission electron microscopy (Carl Zeiss, Germany) [21].On a carbon-coated copper grid, 20 µl of the SPLN formulation were deposited, colored with phosphotungstic dye, and allowed to dry.

Particle size and zeta potential determination
The dispersion, homogeneity, distribution, and, ultimately, the ability to target the particles are all influenced by their size and polydispersity index (PDI) [17].Determining the zeta potential of the SPLN formulation allowed for an assessment of its electrostatic charge, surface characteristics, and stability [19,22].In order to measure the particle size, PDI, and zeta potential in three replicates, we diluted 1 ml of each SPLN formulation with 9 ml of distilled water and used dynamic light scattering (DLS, Germany) to analyze the results [21].

Alzheimer's disease induction
In this study, we used 40 male Wistar rats weighing between 120 and 150 g.Animals were housed in a well-ventilated room in plastic shoebox cages (70 × 35 × 20 cm) and allowed to accommodate for 7 days before starting the experiment.There was an unlimited supply of clean, fresh water and commercially prepared meals.The temperature range was 21-24.5 °C, and the range of relative humidity was 46-60%.

A R T I C L E
The lighting system was maintained using a reversed 12-h light-dark cycle.To make aluminum chloride, the powder was mixed with sterile water and stored in a dark bottle until it was ready to be taken orally.Alzheimer's disease was artificially induced by orally administering AlCl 3 (100 mg/kg) to each rat [23,24].All experiments in this work were performed in in accordance with the rules of the local ethical committee IACUC at the Faculty of Pharmacy, Beni-Suef University.The ethical committee approval number to the study is IACUC, BSU, (022-366).

Experimental design
The rats were split up into four groups of ten at random, as detailed below: A: control negative group; a daily oral gavage of distilled water was used on each rat (neither AlCl 3 nor treatments were obtained).
B: AlCl 3 group (control positive group); a daily oral gavage of AlCl 3 (100 mg/kg) was used on each rat for 4 weeks.
C: AlCl 3 /SP group; a daily oral gavage of SP (300 mg/ kg b.wt) was used on each rat before AlCl 3 administration by 1 h [25].
D: AlCl 3 /SPLN group; a daily oral gavage of SPLN (300 mg/ kg b.wt) was used on each rat before AlCl 3 administration by 1 h.

Behavioral tests
Deficits in learning and memory in rats were evaluated using the novel object recognition (NOR) test.It relies on the rats' innate propensity to explore and investigate novel objects rather than rely on cues from previously encountered objects [26].During the test days, a digital camera was utilized to record the computed parameters.The first part of the test (the familiarization phase) involved placing two known objects in the middle of the arena and leaving the rats there for 10 min.In the second phase (the test phase), which was conducted after 24 h, each rat spent 5 min with one familiar object and one novel object.All of the testing equipment, procedures, and calculation equations were carried out as per the detailed instructions given here [26][27][28].
To evaluate participants' spatial short-term working memory, we used the Y-maze to record their impromptu responses in each arm of the maze [29].All of the testing equipment, procedures, and calculation equations were carried out as per the detailed instructions given here [30][31][32].Throughout the test days, the estimated parameters were videotaped using a digital camera for recording.Each rat was put at the beginning of the (A) arm and left there for 8 min.The other two arms were labeled (B) and (C), respectively, as a result of calculating the order of arm entries in overlapping triplet sets (i.e., ABCCBAABC).
After the behavioral tests are finished, an intraperitoneal (IP) injection of a mixture (0.1 mg/100 g) of ketamine (90 mg/kg) and xylazine (5 mg/kg) at a ratio of 1:1 was administered to each rat for anesthesia.Rats were euthanized via cervical dislocation, and their brains were removed.The brain was dissected into three samples.For the purpose of determining oxidative stress markers, the first sample was homogenized in 5 ml of phosphate-buffered saline for 20 min using a homogenizer (Yellow line DI 18 basic, Deutschland, Germany), and the supernatant was collected.The second sample was placed at − 80 °C in a collection tube of saline, RNase, and protease inhibitors (Promega Corporation, USA) to be used in the determination of gene expression.For the histological analysis, the third sample was washed in saline and then submerged in Bouin's solution.

Determination of oxidative stress markers
Malondialdehyde (MDA) and total antioxidant capacity (TAC) levels in the brain were evaluated in tissue homogenates using particular rat colorimetric test kits [33].The MDA kit was purchased from Egypt's Biodiagnostic Company (Catalogue Number: MD 25 29).The test is based on a 30-min reaction between thiobarbituric acid (TBA) and MDA at 95 °C in an acidic medium.As a result of this reaction, the thiobarbituric acid reactive product was produced.The resulting colored substance's absorbance was measured at 534 nm.The Quan-tiChrom™ Antioxidant Assay Kit was used to determine TAC levels (Catalogue Number: DTAC-100, Hayward, CA 94,545, USA).TAC is measured using Bio-Assay Systems' enhanced assay, which reduces Cu 2+ to Cu + using an antioxidant.Cu + reacts with a dye reagent to produce a colorful product.The TAC of the sample is proportional to the color intensity at 570 nm.

Determination of acetylcholinesterase activity, brain monoamines, and acetylcholine levels
The levels of acetylcholinesterase (AchE) enzyme activity in brain homogenates were measured using a commercially available colorimetric assay kit (BioVision Co., Milpitas, CA, USA).Acetylcholine was determined using HPLC [34].In 75% aqueous HPLC grade methanol (10% w/v), we weighed and homogenized each piece of brain tissue then reconstituted to 1 to 1 volume [35].After a 10-min spin at 4000 rpm, the homogenate's supernatant was frozen at − 80 °C for later use.To detect monoamines in the brain, we used the HPLC technique reported by Pagel et al. [36].The HPLC setup included a quaternary pump, column oven, Rheodyne injector, 20-µl loop, and UV-Vis-NIR variable wavelength detector.The chromatogram and report were generated by a Chemstationpurchased data acquisition application.Solid-phase extraction Chromabond column NH 2 phase cat.No.730031 was used on the sample right away to remove all of the lipids and trace elements.Following this, the material was injected straight into an AQUA 150 × 5 mm C18 column from Phenomenex, USA, with a mobile phase composed of 20 mM potassium phosphate (pH 2.5) at a flow rate of 1.5 ml/min and a UV of 270 nm.After only 10 min, noradrenaline, dopamine, DOPAC, and serotonin were isolated.The ensuing chromatogram showed where each monoamine was located and how concentrated it was relative to the standard, allowing for an accurate calculation of how much of each monoamine was present in the brain tissue.

Gene expression
We used an RNeasy Mini Kit (Qiagen Cato./ID 74,104) to extract total RNA from brain tissue.SuperScript Reverse Transcriptases were used for first-strand cDNA synthesis [37].For quantitative PCR, an ABI Prism StepOnePlus real-time PCR system with PowerTrack™ SYBR Green Master Mix Applied Biosystems™ (Applied Biosystems) was used [38].The primer sets of the examined genes are compiled in Table 1.Following normalization of the target mRNA expression to ACTB, the following was determined: ΔCt = Ct (gene of interest) − Ct (housekeeping gene); ΔΔCt = ΔCt (treated sample) − ΔCt (untreated sample).

Histopathological examination
After being submerged in Bouin's solution, samples of brain tissue were dehydrated in progressively stronger concentrations of ethyl alcohol, cleared in xylol, impregnated with soft paraffin, and finally embedded in hard paraffin.Sections of 5-7 µm were cut and mounted on clear and dry glass slides.Histopathological analysis was performed using a LEICA (DFC290 HD system digital camera, Heerbrugg, Switzerland) attached to a light microscope, and the obtained slides were stained with hematoxylin and eosin (H&E) and Bielschowsky's silver stain [39].The formation of amyloid plaques around neurons in the AD were observed by Congo red stain [40].

Statistical analysis
One-way analysis of variance (ANOVA) and the Tukey test were used to analyze the data using SPSS version 22.
The Tukey test is a post hoc test commonly used to assess the significance of differences between pairs of group means [41].It was used to determine whether the treatment group significantly differed from the control group or not.The data were presented as mean standard error of the mean, and they were statistically significant at the P < 0.05 level.

Results and discussion
Several neuroprotective benefits of SP in various brain regions have been emphasized in numerous in vivo investigations utilizing various animal models [42,43].It was speculated that Spirulina platensis water extract could be used to treat dementia and thus avoid cognitive decline and memory loss, as well as increasing antioxidant activity.Microglial activation is at least largely responsible for the onset of neurodegeneration, and SP treatment has been demonstrated to be neuroprotective.Together, the results show that SP can protect brain cells from damage by blocking or reversing a variety of inflammatory and oxidative neurotoxic pathways at the cellular and molecular levels.In the present study, we, therefore, explore this substance's ability to protect rats from AD as well as its mode of action.

Preparation and in vitro characterization of Spirulina-loaded niosome formulation
Optimum SP-loaded niosome formulation (SPLN) for in vitro and in vivo characterization was chosen after a literature review [15,22,44,45].Based on literature reviews, Tween 60 was chosen as a non-ionic surfactant because its long alkyl chains enabled the creation of small niosomes with a high percentage of EE and a stiff vesicular membrane [14,19,45,46].The high Tween 60's HLB and surface free energy were made for better encapsulation of SP due to its hydrophilicity [14,17,19,45].The results matched those of Waddad et al. and Nowroozi et al. [17,45].Stable niosomal vesicles with high entrapment efficiency are generated in the presence of cholesterol [14,17,19,45].In addition to lowering the surface free energy, cholesterol also increases the hydrophobicity and stiffness of the bilayer.An increase in %EE and a reduction in particle size were seen in conjunction with a Tween 60/cholesterol ratio of 1:2 [14,17,45,47].
The results matched those of Chaw et al. and Waddad et al. [22,45].As a charge inducer, DDP is employed to help ensure that the niosomes are being prepared to have a highly negative zeta potential value, which is good for their stability [19,45].According to literature reviews, the optimum formulation included Tween 60, cholesterol, and DDP at the following molar ratios: 1:2:0.1 [14, 17,

Behavioral test
In Fig. 2, a significant (P < 0.01) decrease in the discrimination ratio (DR) (Fig. 2A), the discrimination index (DI) (Fig. 2B), and the difference score (DS) (Fig. 2C) was observed in the control positive group compared to the control negative group.
Treatments with SP and SPLN formulation improved recognition memory significantly (P < 0.01) by increasing DR, DI, and DS when compared to the control positive group.Additionally, the SPLN formulation significantly increased DR (P < 0.05) in comparison with SP.
Figure 3 shows that the control positive group saw a statistically significant (P < 0.01) reduction in spontaneous alternation behavior (SAP) as compared to the control negative group.When compared to the control positive group, treatment with free SP and SPLN formulation significantly improved the impaired working memory by increasing SAP (P < 0.01).Our obtained data clarified that AlCl 3 plays a crucial role in AD modeling as it can cross the blood-brain barrier, causing neuron loss, amyloid plaque precipitation, and memory impairment.

Determination of oxidative stress markers
As shown in Fig. 4, AlCl 3 caused significant (P < 0.01) oxidative stress in the brain by decreasing the antioxidant marker (TAC, Fig. 4A) while increasing the pro-oxidant marker (MDA, Fig. 4B) compared to the control negative group.Treatments with free SP and SPLN formulation significantly (P < 0.01) improved brain oxidative status by decreasing MDA and increasing TAC in comparison with the control positive group.Additionally, SPLN formulation significantly (P < 0.001) increased TAC, decreased MDA, and switched the total antioxidant capacity from deviation to control levels in comparison with free SP because niosomes improved SP's permeability, localized and stored it in the brain, and then slowly released the medication over time.Our obtained data clarified that AlCl 3 as a neurotoxin plays a crucial role in AD modeling, causing oxidative stress by increasing MDA and decreasing TAC.Our results were in line with prior research, which demonstrated a significant rise in brain MDA, while enzymatic antioxidant expression was significantly reduced in the aluminum-treated animals [53,54].Treatment with free SP and SPLN formulation alleviated the oxidative stress in the brain.Our results were in line with prior research, which observed that SP has been shown to have antioxidant potential due to the presence of bioactive components such as polysaccharides and carotenoids, which have high antioxidant properties [55].Exposure of rats to a strength training regimen revealed marked oxidative stress, with elevated MDA and decreased TAC, which was significantly alleviated by SP [56].

Determination of acetylcholinesterase activity, brain monoamines, and acetylcholine levels
The results in Fig. 5 demonstrated that the AchE activity (Fig. 5A) significantly (P < 0.05) decreased and the Ach level (Fig. 5B) significantly (P < 0.05) increased in the control positive group compared to the control negative group.In comparison to the control positive group, treatments with free SP and SPLN formulation significantly (P < 0.01) restored AchE activity and Ach to the normal levels.Our obtained data clarified that AlCl 3 plays a crucial role in AD modeling by disturbing brain neurotransmitters.Our results were in line with prior research, which demonstrated a significant decline in AchE activity [50,57].Increased Ach level may be attributed to decreased AchE activity, which prevents circulating Ach from being degraded L E [58,59].Furthermore, AlCl 3 resulted in a substantial decline in hippocampal monoaminergic neurotransmitters, mainly dopamine, serotonin, and norepinephrine [6,60].Additionally, the antiapoptotic protein (Bcl-2) levels and Mao transcript considerably decreased, and the proapoptotic (Bax) levels increased in the AlCl 3 -treated group [61].Moreover, Ibrahim et al. reported that the reduced brain neurotransmitters and downregulation of AchE and Mao resulted from brain neurotoxicity [62].Aluminum, a metal ion of toxicological relevance, inhibits the activities of Na + , K + -activated, and Mg 2+ -activated adenosine triphosphatase [63].Compared to the control positive group, treatments with free SP significantly (P < 0.05) increased the reduced AchE activity and upregulated the AchE.The results matched those of Fathy et al. and Galal et al. [64,65].
Figure 6 shows that the activity of monoamines (NE, 5HT, DA, and DOPAC) in the brain was significantly (P < 0.05) lower in the control positive group compared to the control negative group.Treatments with free SP and SPLN formulation restored the brain monoamines' activity significantly (P < 0.01) in comparison to the control positive group.Our obtained data clarified that aluminum neurotoxicity increases the levels of neopterin in the brains of AD patients while also resulting in a decrease in brain neurotransmitters including dopamine, norepinephrine, and serotonin by decreasing the concentrations of tetrahydrobiopterin in the cerebrospinal fluid, which is necessary for the production of such neurotransmitters.The results matched those of Foster et al. [66].Treatment with SP and SPLN ameliorated the deleterious effect of AlCl 3 on brain monoamines.Fathy et al. and Galal et al. recorded a significant increase in brain monoamines levels by SP treatment in rats [64,65].

Gene expression
Bcl-2 expression has a significant impact on neuronal development during embryogenesis, with Bcl-2 deficient mouse embryos exhibiting significant neuronal development defects [67].Overexpression of Bcl-2 stimulates DNA repair after DNA damage caused by the oxidative stress.Figure 7 summarizes the gene expression data, revealing an increase in Bax mRNA levels and a decrease in bcl-2, AchE, and Mao mRNA levels in the control positive group compared to the control negative group.These results matched those The nerve cells and neuroglia showed severe degenerative changes.Accumulation of acidophilic masses (arrowhead).A3: the SP-treated group showed meningeal membrane suffering from edema (E).The cellular layers of hippocampus appeared within normal range with few degenerated neurons and neuroglia cells.A4: the SPLN-treated group showed normal tissue of hippocampus.H&E stain × 200.B1: the control negative group showed the normal architecture of the hippocampus with normal neurons (arrow) as well as normal neuroglia (arrowhead).B2: the control positive group; the hippocampus appeared with few layers.The nerve cells (arrow) and neuroglia cells (arrowhead) showed severe degenerative changes with shrinkage.B3: the SP-treated group showed that the hippocampus was regaining its layers architecture.The majority of nerve cells (arrow) and neuroglial tissue (arrowhead) appeared normal except few cells appeared degenerated (double arrow).B4: the SPLN-treated group revealed normal architecture of the hippocampus containing normal neurons (arrow) and normal neuroglia cells (arrowhead).H&E stain × 400 Fig. 9 Histopathological evaluation (Congo red and silver stain).C1: the control negative normal group showed that the hippocampus appeared normal without any foreign materials accumulation.C2: control positive (AlCl 3 -treated group) showed that a marked deposition of acidophilic amyloid materials in the hippocampus in between (arrow) and/or inside (arrowhead) the neurons.C3: the SPtreated group showed acidophilic materials between the neurons (arrow) and little droplets inside the nerve cells (arrowhead) of the hippocampus.C4: the SPLN-treated group revealed that the hippocampus appeared as a normal group without any amyloid deposi-tion.Congo red stain × 400.D1: the control negative group showed that the tissue of the hippocampus containing highly branched nerve fibers (arrow) with many spots of synapse (arrowhead).D2: control positive (AlCl 3 -treated group) showed that the nerve fibers of the hippocampus appeared less branched with rare spots of synapse.D3: the SP-treated group revealed that the tissue of hippocampus containing nerve fibers with moderate branching (arrow) with points of synapse (arrowhead).D4: the SPLN-treated group showed the nerve fibers of hippocampus appeared as normal with highly branched neurons (arrow) with many spots of synapse (arrowhead).Silver stain × 200 and other neurotoxins caused a significant downregulation of MAO and AchE as a result of brain neurotoxicity [62,68].Treatments with free SP and SPLN formulation ameliorated the injurious effects of the AlCl 3 .Furthermore, when compared to free SP, SPLN formulation significantly (P < 0.01) upregulated their gene expression because niosomes improved SP permeability, localized and stored it in the brain, and then slowly released the medication over time.SP treatment significantly increased AchE activity as well as brain monoamine levels in rats, according to Fathy et al. and Galal et al. [64,65].

Histopathological examination
The normal tissue of the hippocampus was covered by a fine meningeal layer rich with blood vessels.The architecture of the hippocampus appeared normal, with well-demarcated layers of neurons with active nuclei, basophilic cytoplasm, and well-demarcated neuronal processes, as well as normal neuroglia enclosing the nerve cells (Fig. 8, A1 and B1).When stained with Congo red, the hippocampus appeared normal, with no foreign material accumulation.The hippocampus contains highly branched nerve fibers with many spots of synapse (Fig. 9, C1 and D1).The male rats treated with aluminum chloride showed severe tissue damage in the hippocampus in the form of severe fibrous thickening with prominent edema and highly congested blood vessels in the meninges covering the hippocampus.The nerve cells and neuroglial tissue appeared as a few disrupted layers and exhibited severe degenerative changes with massive cell shrinkage as well as marked vacuoles all over the brain tissue (Fig. 8, A2 and B2).Sections of the hippocampus stained with Congo red stains showed deposition of acidophilic amyloid materials in between and/or inside the neurons.The nerve fibers of the hippocampus appeared less branched with rare spots of synapse (Fig. 9, C2 and D2).Buraimoh et al. and Kamel et al. found that rats exposed to oral AlCl 3 administration showed neuronal degeneration in various hippocampal areas [69,70].Interestingly, Congo red staining confirmed a slight positive reactivity of amyloid plaques that could be seen in the brains of AlCl 3 -treated rats [6].The brain tissue in these rats was protected from the harmful action of AlCl 3 by administration of SP and SPLN, with the tissue of the hippocampus appearing more or less normal in these groups, respectively (Fig. 8, A3,4 and B3,4; Fig. 9, C3,4 and D3,4), which is supported by Galal et al. and Yousef et al., who discovered that SP treatment improved and reduced neurodegenerative changes in the brain [56,65].
Novel therapeutics for Alzheimer's disease management, as well as symptom-reducing medications for cognitive decline and neuropsychiatric symptoms, are urgently needed.Our research has looked into the effectiveness of SP and SPLN in alleviating Alzheimer's disease in rats and mice.Surprisingly, SP and SPLN significantly reduced the negative effects of AlCl 3 via ameliorating cognitive dysfunction, reducing oxidative stress, restoring brain neurotransmitters, upregulating gene expression, and playing a neuroprotective role in the brain.

Conclusion
Aluminum-induced AD is one of the main risk factors for dementia all over the world.Spirulina platensis (SP) has shown anti-inflammatory and antioxidant abilities by reducing oxidative stress, free radicals, and other reactive oxygen species.The purpose of our research was to deliver SP in a controlled and targeted manner using niosomes in order to improve the effectiveness and selectivity of SP as an AD therapy.Different SP-loaded niosome (SPLN) formulations were prepared and evaluated to choose the optimum formulation for further studies such as the efficacy and toxicity of SPLN in comparison to oral SP using the AlCl 3 -induced AD rat model.Compared with the AlCl 3 control group, rats treated with the SPLN formulation showed antioxidant, anti-neuroinflammatory, and cholinergic signaling enhancements.The SPLN formulation enhanced recognition and working memories and restored the reduced brain neurotransmitters.In conclusion, the SPLN formulation could be considered an effective AD therapy.
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Fig. 1 Fig. 2 Fig. 3
Fig. 1 Transmission electron microscopy of SPLN formulation R E T R A C T E D A R T I C L E

Fig. 4 R
Fig. 4 Effect of SPLN formulation on TAC and MDA levels in rat brain.Results are expressed as means ± SE.The different superscript indicates significant difference at P < 0.001 Figure3shows that the control positive group saw a statistically significant (P < 0.01) reduction in spontaneous alternation behavior (SAP) as compared to the control negative group.When compared to the control positive group, treatment with free SP and SPLN formulation significantly improved the impaired working memory by increasing SAP (P < 0.01).Our obtained data clarified that AlCl 3 plays a crucial role in AD modeling as it can cross the blood-brain barrier, causing neuron loss, amyloid plaque precipitation, and memory impairment.Consistent with the findings of Singh et al. and Mohamed et al., we discovered that oral treatment of AlCl 3 decreased working memory in the y-maze test and short-term memory in the NOR test in AD model rats [49, 50].Treatment with SP and SPLN formulation improved the impaired recognition memory and spontaneous alternation behavior.Consistent with previous studies by Wang et al. and Imai et al., we found that SP treatment enhanced DI in the novel object test and enhanced spontaneous alternation behavior in the Y-maze in an AD rat model [51, 52].Additionally, SPLN formulation significantly increased recognition memory and spontaneous alternation behavior in comparison with SP because niosomes improved SP's permeability, localized and stored it in the brain, and then slowly released the medication over time.

Fig. 5 R
Fig. 5 Effect of SPLN formulation on AchE and Ach levels in rat brain.Results are expressed as means ± SE.The different superscript indicates a significant difference at P < 0.05 and 0.01

Fig. 6 R
Fig. 6 Effect of SPLN on monoamines levels (NE, 5HT, DA, DOPAC) in rat brain.Results are expressed as means ± SE.The different superscript indicates a significant difference at P < 0.05 and 0.01

Fig. 7 Fig. 8
Fig. 7 Effect of SPLN against AlCl 3 -induced alterations on brain mRNA expression of A AchE, B Mao, C Bcl-2, and D Bax.Data are presented as mean ± SE (n = 6).The different superscript indicates a significant difference

of
Elmorsy et al. and Ibrahim et al., who found that AlCl 3

Table 1
The primer sets of the assessed genes