Alzheimer’s disease (AD) is one of the furthermost advanced neurodegenerative disorders in persons beyond 65 years of age producing loss of neurons and finally dementia.1 It is caused due to neurodevelopmental disorders that result from an imbalance between excitatory and inhibitory signaling of the hippocampus and the cerebral cortex.2 The extracellular precipitation of amyloid-beta (Aβ1-42) and the neurofibrillary tangles of hyper-phosphorylated tau proteins are intellect to be the two vital pathologic hallmarks of AD,3 associated with stimulating acetylcholine esterase (AChE) enzyme to form insoluble Aβ plaques, induced neural cell death.4,5 Consequently, these pathological neural structure changes result in memory loss and cognitive dysfunction in AD patients.6
However, the extracellular matrix (ECM) is a crucial key regulator for synapse formation and regulation of its function in the cerebral cortex.7 When damage or alteration of the matrix arrangement occurs, it implicitly modulates neuronal activity, synaptic plasticity, and amyloid beta-42-A aggregation in AD.8 Hyaluronic acid (HA) is a crucial component of the central nervous system and a scaffold component of the extracellular matrix (CNS).9 It also modulates intercellular signaling through interactions with Hyaluronan neuronal cell surface receptors like Cluster of Differentiation 44 (CD44), Hyaluronan-mediated motility (RHAMM), and Intercellular adhesion molecule 1 (ICAM).10 The degradation of hyaluronic acid as a result of an excessive inflammatory response brought on by tissue trauma and neurodegenerative disease leads to the remodeling of ECM of the nervous system.11 The NF-kB pathway can be regulated by miRNA-137, which inhibits pro-inflammatory responses by targeting ICAM-1 and CD44, and tumor necrosis factor-alpha-Induced Protein 1 (TNFAIP1), inhibiting the neurodegenerative disorders of AD. The NF-kB pathway is also inhibited when miRNA-137 is overexpressed in neurons.12
Otherwise, the main cogitation centre was the central cholinergic system.13 Acetylcholine (ACh) is the primary cholinergic neurotransmitter synthesis in the central neurons responsible for maintaining consciousness, recognition, thinking, and learning, especially in the basal forebrain, frontal lobe, hippocampus, and brainstem.14,15 Decreased Ach levels in the brain significantly characterize AD; neurodegenerative dysfunction symptoms appear.15
Citicoline sodium (CIT) is a choline supplier, which is a substantial intermediate in the synthesis of phosphatidylcholine (indispensable brain phospholipids).16 The neuroprotective drug CIT is therapeutically used to treat conditions such as Alzheimer’s disease (AD), Parkinson’s disease, stroke, and brain ischemia.16 Citicoline can benefit both degenerative and vascular cognitive incline in an assortment of ways including (apoptosis suppression, neuroplasticity reinforcement, phospholipid, and acetylcholine ACh synthesis).17 However, when CIT was taken orally or parenterally, it quickly converts to cytidine and choline in a few minutes.18 These two separate metabolites are consumed by various parts of the body and then reach the brain. Even though the absorption of the drug is very high with a bioavailability of over 90%, the percentage drug delivered to the brain is very low, only 0.5% and 2% when taken orally or injected, respectively.19 This may be clarified by the strong polarity of CIT and its fast metabolism followed by liver uptake of most of the free choline which represents the fundamental obstacle hindering intact drug molecules from crossing the blood–brain barrier.20 These demands necessitate the development of an appropriate delivery nanocarrier to target the CIT directly to the brain and realize its therapeutic profit in AD.
One of the nanocarriers for the transport of active compounds that is now being explored the most frequently is the liposome. They are vesicular structures with a spherical form that range in size from 25 nm to a few microns and are made of phospholipids with a hydrophilic head and two hydrophobic tails.21,22 There are many benefits of using liposomes as a drug delivery vehicle, including higher efficacy and bioavailability of encapsulated substances, high biodegradability and biocompatibility with low toxicity, ability to self-assemble, the encapsulation of both hydrophobic and hydrophilic agents, and possibility of facile removal from the body.23,24 Additionally, these vesicular nanostructures enable the modification of important physicochemical characteristics, such as size and surface. The ability to change important physicochemical characteristics, such as size and surface, is another feature of these vesicular nanostructures that is essential in the application.25 Liposomes have been utilized in the food, cosmetic, agricultural, and, most significantly, pharmaceutical industries since the 1960s.26 As indicated by the fact that liposomes were the first nanoscopic drug delivery technology to be approved for clinical use (Doxil®, in 1995),27 the development of this type of nanocarrier is really very dynamic and prospective.
Liposomes’ insufficient chemical (hydrolysis of ester linkages or peroxidation of the unsaturated acyl chain) and physical (destabilisation processes such agglomeration, flocculation, or coalescence, which ultimately lead to changes in the size of the nanostructure) stability limit their application.28 Novel structural liposomes were created using supportive additives like cholesterol, ethanol, or non-ionic surfactants to improve the stability and efficacy of drug loading in conventional vesicle vehicles.
The second class of liposomes made of phospholipids and additional single-chain surfactants, also known as edge activators, are called transferosomes.29 The use of transferosomes as a drug delivery vehicle has a variety of advantages, including its capacity to accommodate therapeutic molecules with a variety of solubilities due to their infrastructure, which combines hydrophobic and hydrophilic moieties. They can swell and squeeze through a tiny hole (between 5 and 10 times smaller than their own diameter) without suffering a substantial loss. Drugs of both low and high molecular weights, such as analgesics, anaesthetics, corticosteroids, sex hormone, anticancer, insulin, and albumin, can be transported via them. Protein and peptides are two examples of how they protect the medicine from metabolic deterioration. They serve as depots and release their contents gradually.30,31 They can be utilized for both systemic and topical medication delivery. Because the technique is uncomplicated and avoids needless use of pharmaceutically undesirable ingredients, they are simple to scale up. That on first look, transfersomes resemble liposomes, a type of lipid bilayered vesicle. However, in terms of functionality, transfersomes are significantly more malleable and flexible than routinely employed liposomes. The fundamental drawback of transferosomes is that they are prone to oxidative destruction, which makes them chemically unstable.30
The most recent literature studies describe bilosomes, the next generation of “soft” lipid vesicular nanocarriers, in which bile salts such as deoxycholic acid, sodium cholate, or sorbitan tristearate operate as edge activators. Comparing the system to the traditional liposome and transferosomes, the addition of these biosurfactants boosts the colloidal stability of the system.32 Bilosomes (BLs), which are mostly made of nonionic surfactants and bile salts, are a new approach to vesicular drug delivery.33,34 They are metastable in membrane organization due to the incorporation of bile salt molecules that decrease the phase transition temperature producing them ultra-flexible and highly deformable under the physiological temperature.35 Bilosomes have been applied for transdermal,36 topical,37 and ocular drug delivery.34
However, no more published studies have examined the impact of intranasal administration of medication-loaded BLs on absorption of drug and brain targeting. Although BL vesicles have not been considerably discussed for intranasal medicine delivery, their unique small size, incorporated with the bile salts in their constitution, supplies promising scenarios for using them in nose-to-brain drug delivery.
The theory of brain drug targeting using nose dosage form in the cure of neurodegenerative disorders has profited a great deal from nanoscience, provided that nanovesicles are capable of overcoming the fast mucociliary clearance and poor nasal absorption.38 Intranasal delivery of nanovesicles offers various advantages, such as ease of self-administration, non-invasiveness as well as successful brain targeting.39 Despite the multiple advantages of bilosomal vesicles, BL suitability as a systemic delivery vehicle is compromised owing to a lack of specificity and easy sequestration by the reticuloendothelial system.40
To get over these restrictions, these unique vesicles were created and fabricated by combining Span 60, Phospholipon 90 G, sodium deoxycholate, and cholesterol in various ratios to boost the rate of drug loading and enhance the vesicle’s ability to penetrate biological membranes; the resulting vesicle was given the name Transbilosome.
Ultra-flexible transbilosomes (TBLs) are an unique permeation-enhancing lipid nanovesicular system derived from bilosomes, which were initially defined by Conacher et al,41 and from transferosomes, which Cevc and Blume established in 1992.29 TBLs are elastic, malleable lipid vesicles that contain a variety of ratios of phospholipids, Span 60, cholesterol, and sodium deoxycholate. They effectively penetrate biological membranes, particularly nasal mucosa, due to their tiny particle size, high entrapment efficiency percentage, high in vitro release, increased permeability, and increased permeation. TBLs are thought to be superior to transferosomes and bilosomes for drug delivery through the intranasal mucosa. Additionally, these unique vesicles were fabricated with an aim to create transbilosomes by combining the benefits of transfersomes and standard bilosomes in a single formula. Additionally, nanovesicle surface engineering becomes very important to potentiate and attenuate a potential payload.
This study was carried out to investigate the prospective role of implementing (HA), a main component of the ECM, as the natural legend and a surface-bound targeting moiety (RHAMM), CD44, and ICAM for neural cell surface receptors in brain cells, facilitating special uptake and persuasive therapeutic efficacy.10,42 Thus, HA-decorated transbilosomes (TBLs) were developed and fabricated to enhance CIT permeation across nasal mucosa, distribution, controlled-release characteristics, specific brain targeting, and modulation of mucoadhesion of the coated formula by further incorporation of mucoadhesive and thermosensitive gelling system with extensive nasal mucociliary transit time. A temperature-induced in situ gelling system that turns into a gel appearance at body temperature was fabricated utilizing poloxamer 407 and poloxamer 188 combined with mucoadhesive polymer Carbopol 971 to boost the intranasal residence time of the thermogelling system.
It was found that the earlier literature lacks satisfactory data about any targeting CIT delivery to the brain. Thus, this study was established to achieve two main targets. The first one was to formulate CIT-HA*TBLs thermogel for intranasal delivery of CIT to the brain. The Second target was to assess the in vivo efficiency of the CIT-HA*TBLs thermogel compared to (CIT solution, control thermogel, UN hyuloranated CIT-TBLs, and HA-TBLs) using a rat model of AlCl3-induced AD.
Citicoline sodium (CIT) was obtained as a kind gift from GLOPAL NAPI Pharmaceutical (6th of October City, Egypt). Lipoid AG, (PHOSPHOLIPID GmbH Nattermannallee1 Sennweidstrasse44, CH-6312Steinhausen, and Switzerland) kindly supplied Phospholipon 90 G (PL 90 G). The following items were bought from Sigma-Aldrich (St. Louis, MO, USA): cellulosic membrane (12,000–14,000 kDa molecular weight cut-off), sodium deoxycholate (SDC), hyaluronic acid (HA), Poloxamer 407 (PL 407), Poloxamer 188 (PL 188), Carbopol 971 (CAP971), and cholesterol (CHO). Methanol HPLC grades, chloroform and Cetalkonium chloride (CKC), and sorbitan monostearate (Span 60) were purchased from Across Organics (Cairo, Egypt). Aluminum chloride (molecular weight: 133.332 g/mol) was purchased from El-Gomhouria Chemical Company (Cairo, Egypt). The microRNA-137 (miR-137) qRT-PCR Master Mix for a quantitative real-time polymerase chain reaction was obtained from Thermo Fischer Scientific Co., Massachusetts, USA (Cat. No. 4427975). The ELISA kits were purchased for Nuclear Factor Kappa B (NF-kB) and reduced to Malondialdehyde (MDA) from My BioSource Co., San Diego, California, USA (Cat. No. MBS268427 and MBS453975, respectively). The anti- amyloid-beta (Aβ1–42) primary antibody for the immunohistochemical assay was obtained from Thermo Fischer Scientific Co., Massachusetts, USA (Cat. No. BS-0107R). All of the other reagents were of analytical grade.
A 33 Box–Behnken Design Expert® (Version 13, Stat-Ease Inc. Minneapolis, MN) was utilized to prepare 15 formulae of CIT-TBLs where the independent variables were the amount of PL90G (X1), Sp60 (X2), and SDC (X3). According to Table 1, the dependent variables were the following: particle size (Y1), zeta (Y2), EE% (Y3), % Cumulative drug released for 8h (Y4), and Cumulative drug permeated through nasal mucosa for 24h (Y5).
Table 1 The Independent Variables, Their Respective Levels, and the Summarize Statistics Model of Box–Behnken Design Used for Optimization of CIT Transbilosomes
Different CIT-TBLs formulae were prepared according to the technique of thin-film hydration with some modifications.43 Briefly, the mixed lipid consisting of PL 90G, SP60, and constant weight of CHO (10 mg) was added to bile salt and dissolved in 15 mL chloroform: methanol (2:1) in a round-bottom flask. The produced organic solution was evaporated at 60°C for 30 min under reduced pressure using a rotary evaporator (Stuart rotary evaporator, RE300, Wolf Laboratories, North Yorkshire, UK, with Stuart vacuum pump, RE3022C, Wolf Laboratories, North Yorkshire, UK), until a thin, entirely dry film was created. The resulting film was maintained for 2 h in a desiccator under vacuum for complete removal of traces of solvent.
The dried lipid film was then completely hydrated by adding 10 mL of SNES pH 5.5 containing 50 mg CIT and the flask was allowed to rotate at 100 rpm for 1 hour under normal pressure. The suspension was preserved at 4 °C for complete hydration process through phospholipid swelling (maturation), resulting in large multilamellar vesicles.44,45 This step increases the loading of the CIT medication into the vesicle, also keeping the vesicle overnight aids in stabilizing it.46 The size of the multilamellar TBLs was subsequently decreased by sonicating the suspension for 30 minutes in a bath sonicator (Sonix TVs-series ultrasonicator, Sonix IV Ultrasonic Cleaning Systems, North Charleston, SC).47,48
Table 3 details the components of these formulae. The optimized formula was integrated and coated by HA (1.5% w/v),38 in 10 mL of SNES pH 5.5 containing 50 mg CIT, and in the case of coating, the charge inducer (CKC) was requisite for use. For the preparation of the HA formula, CIT-TBLs were decorated with HA by which (CIT-HA*TBLs) formed via electrostatic interaction between positively charged CKC in the organic phase and negatively charged HA in the aqueous phase. The decorated formula was then kept in the refrigerator for further studies.48
Table 2 Primers Sequence of Mi RNA 137 Gene
Table 3 Observed Response in Box–Behnken Experimental Design of CIT-Loaded TBLs
The average size distribution and polydispersity index (PDI) of CIT-TBLs were evaluated by the dynamic light scattering method (DLS) using a Malvern Mastersizer.49,50 Briefly, the TBL samples (0.1 mL) were diluted 100 times using deionized water to obtain an efficient light scattering intensity; then, the temperature was maintained at 25°C with a 90° scattering angle. Determination of surface charge was performed using the same instrument. The approach analyzes the electrophoretic motion of the particles in an electrical field using the same diluted sample.51
The ultracentrifugation technique was applied to determine the EE% of the CIT-TBLs suspension. The samples were kept at 4 °C overnight for maturation of the TBLs vesicle and then centrifuge at 14,000 rpm for 3hrs at 4°C using a refrigerated centrifuge (SIGMA 3–30K, Steinheim Germany).52 The free drug (supernatant) was separated from TBL pellets and the amount of entrapped CIT was analyzed spectrophotometrically for the concentration of CIT (Jasco V-530, Japan) at λmax 270 nm. Drug EE% was calculated according to the following equation:
(1)
The diffusion analysis was performed according to the dialysis method using vertical diffusion Franz cells with an effective diffusion area of 5 cm2.53
TBLs’ suspension of different formulae equivalent to 3mg drug was placed in the donor compartment. A 50mL of SNES pH 5.5 was utilized as a receptor medium.54 A magnetic stir bar was used to agitate the receptor compartment, which was kept at 37°C and 100 rpm. A cellulose dialysis membrane with a molecular weight cutoff of 12,000 kDa that had been soaked in the receptor medium overnight was used to separate the donor compartment from the receptor compartment. To maintain a constant volume, 3 mL aliquots were taken out of the sampling port and replaced with an equivalent volume of fresh medium at regular time intervals (0.5, 1, 2, 3, 4, 5, 6, 7, and 8 h).
The CIT content of the aliquots was analyzed using a “UV spectrophotometer” at a λmax of 270 nm. The analysis was repeated three times and the percent CIT released was expressed as the mean (±SD). The cumulative percent of CIT released from CIT-TBLs was plotted against time. To identify the CIT release pattern, the data were used with various models of release kinetics such as zero-order, first-order, and Higuchi’s matrix.55 Using the following equation:
(2)
The ex vivo permeability studies of CIT-TBLs formulae and CIT solution were performed as described above in the in vitro release study under the same conditions using fresh isolated camel nasal mucosa. The mucosa was attained instantly following animal sacrifice in a nearby slaughterhouse and utilized as a model membrane because its morphology and permeability are similar to those of the human nose.56 The dominant nasal concha was recognized, dissociated from the nasal membrane, and allowed to acclimate for 30 min in PBS pH 6.4.57 Different CIT-TBLs formulae equivalent to (3 mg of CIT) were added to the superior mucosal surface (5cm2) and placed into receptor media. The temperature of the receptor chamber containing 50 mL of PBS pH 6.4 is controlled at 37±0.5°C under continuous stirring with the magnetic bar at 100 rpm, in a way that the surface of nasal mucosa just flushes the diffusion fluid. The experiment was carried out under a non-occlusive state. At regular times, formulae samples were taken out of the receptor compartment (1, 2, 3, 4, 5, 6, 8, 10, 12, and 24 h); then, the receptor chamber was recompensed with equal volumes of fresh milieu. The withdrawn samples were filtered through a 0.45-µm Millipore filter and finally measured at 270 nm using a spectrophotometer. For each formula, the cumulative amount of CIT drug permeated per unit area (μg/cm2) was plotted versus time (h).
The permeation parameters involving Q24 in μg/cm2, the lag time in min, permeability coefficient (KP) in cm/h, and drug flux (Jss) in μg/cm2h were computed for each formula and the control CIT solution. Additionally, the enhancement index (EI) was calculated applying the following equation:
(3)
The optimization process was carried out following three-factor, three-level Box–Behnken design as illustrated in Table 1 using Design Expert® (Version 13.0, Stat-Ease Inc. Minneapolis, MN). A design matrix comprising 15 experimental runs was used in TBLs formulae (Table 3). The amount range and ingredient of independent variables were chosen upon preliminary research experiments and data collected from the literature review. The nonlinear computer-engendered quadratic model was given the following equation:
(4)
where Y is the dependent measured response associated with each factor-level combination; b0 is an intercept; b1, band b3 are regression coefficients;b12,b13, andb23 are interaction coefficients, while b11, b22, and b33 represent interaction and quadratic coefficients computed from the resultant experimental values of response from experimental runs; and X1, X2, and X3 are coded intensity levels of the independent variables. While the amount of all PL90G (X1), Sp60 (X2), and SDC (X3), which is a process independent variable introduced as low (−1), medium (0), and high (+1), as described in Table 1.
The same procedures previously described for the characterization of un-hyaluronate TBLs in the determination of EE%, VS, ZP, PDI, Q8h, and Q24h section were adopted.
The morphology and size of optimized CIT-TBLs and CIT-HA*TBLs were examined using a transmission electron microscope (TEM-HR-2100, Joel, JAPAN).58 The freshly prepared samples were negatively stained with 2% phosphotungstic acid that was previously diluted with double-ionized water and then applied onto a 400-mesh copper grid coated with carbon film. Finally, the samples were left to dry at room temperature to allow adherence of CIT-HA*TBLs and CIT-TBLs formulae to the carbon film. TEM observation was carried out at an accelerating voltage of 200 kV and the photographs were taken at suitable magnifications.40
The thermal properties of the plain drug (CIT), excipients (PL90G, SP 60, SDC, CHO, CKC, and HA) drug–excipients mixture (physical mix in a ratio of 1:1), and the optimized CIT-HA*TBLs formula were assessed using DSC (DSC 50 Shimadzu, Kyoto, Japan). First, 5 mg samples were placed, and hermetically sealed in flat crucible aluminum pans, and then they were heated at 25–400 °C at a scanning rate of 10 °C/min under inert nitrogen flow at 25 mL/min.32 DSC thermograms were recorded.59
The optimized CIT-HA*TBLs were stored for 30, 60, and 90 days in a glass vial at 4 °C, and then the VS, Zp, and EE% were assessed.60
A thermosensitive gel system was created using the optimized CIT-HA*TBLs formula. Poloxamer 407 (22% w/v), Poloxamer 188 (10% w/v), and Carbopol 971 (1% w/v), were all included in the thermogel base combination.61 The cold procedure, as previously described, was used to create the gel base.62,63 An accurately weighed amount of the three polymers, and preservatives (mixture of 0.1% methyl paraben and 0.01% propyl paraben) were sprinkled in the calculated amount of CIT–HA*TBLs (5 mg CIT/g). The mixture was stirred using a magnetic stirrer to ensure complete polymers’ solubility at room temperature until no lumps were observed then stored overnight at 4°C to ensure complete swelling and form in situ gel. The previous technique was applied to form control in situ gel (free CIT without HA*TBLs vesicles) by sprinkling the polymeric ingredients on a solution of CIT dissolved in SNSS.
The sol-to-gel phase transition temperature (gelation temperature) was measured for all the prepared CIT-HA*TBLs thermogel formulae according to the technique described previously by Mansour et al.64 An aliquot of 2 mL of the primed dispersion was transported to a test tube in a thermostatically digital water bath at 15°C and wrapped well by the parafilm membrane. The temperature of the bath was elevated in increments of 3°C (or 0.5°C in the region of Sol–gel) and left to equilibrate at each new setting. The thermogel samples were examined for gelation, which was thought to have occurred when the meniscus would no longer move upon orientated to 90°. All measurements were done in triplicates.
The rheological properties of the produced thermogel formulae were determined utilizing Cone and Plate programmable viscometer (DV-III Ultra viscometer, RV model, Brookfield, USA). A sample (0.5 gm) of the tested formula was applied to the lower plate of the viscometer. The viscosities were measured at two different temperatures 25±0.5°C and 37±0.5°C by circulating bath connected to the viscometer-using spindle 52 at a shear rate ranging from 20 to 200 S−1. The viscosities, as well as the area of hysteresis loops, were determined. Using the trapezoidal method, the area of the hysteresis loops was calculated. The area under the lower curve was then subtracted from the area under the upper curve. Farrow’s equation was applied to examine how different gel bases flowed.
(5)
To determine the value of N, which represents the departure from Newtonian law, log G was plotted against log F. If N is smaller than 1, dilatant flow is present (shear rate thickening).65
A 0.5 gm of intranasal thermogel and control gel (equivalent to 5 mg CIT) were utilized in the in vitro release and the ex vivo permeation studies using the same procedure as described above.
The Ethical Review Board of the Faculty of Pharmacy at South Valley University, Qena, Egypt (acceptance D20-21) permitted the experimental process. The experiment was performed on adult male Wistar rats weighted 180–200 g. Rats were maintained in an air-conditioned (25 ± 1°C) pathogen-controlled animal room for 2 weeks for adaption before being subjected to laboratory tests with free access to standard forage and tap water ad libitum. The study followed the National Institute of Health’s (NIH) Guide for the Care and Use of Laboratory Animals (Publication 85–23, modified 1985) guidelines.
The experiment was carried out on seven weight-matched groups consisting of 8–10 rats. The first group was maintained as a standard normal control received only vehicle tween 80. The second group was kept as a positive control group and received aluminum chloride (AlCl3) intra-peritoneal for 6 weeks in dose (100 mg/kg/i.p).66,67 Treatment groups received intranasal-tested agents the CIT solution and the freshly prepared thermogel as follows: Group 3 received a CIT solution, Group 4 received CIT control thermogel, Group 5 received CIT-TBLs thermogel, Group 6 received CIT-HA*TBLs thermogel, and Group 7 received HA*TBLs thermogel all treatment received for consecutive 2 weeks in dose (0.9mg/kg).68,69 The doses of test agents were determined with pilot trials guided by the published literature. For the intranasal application of thermogel, rats were held horizontally at a 45-degree angle. Then, the animal’s back was supported by the palm of the manipulator’s hand, and the skinfold of the neck was fixed between the thumb and the index finger. The thermogel was administered with a micropipette as a small drop covering both nostrils at the end of administration, the rats were returned to their home cage.70–72
At the end of the experiment, animals were sacrificed via cervical dislocation followed by separation of the brain immediately, and hippocampus excision was then cleaned with iced saline for biochemical, histopathological, and immunohistopathological examination (Figure 1).
Figure 1 Experimental design of in vivo study.
Rats were sacrificed by cervical dislocation, the skull was opened, and the brain of each rat was promptly extracted and cut mid-sagittal into two hemispheres. One cerebral cortex hemisphere was cleansed with ice-cold saline to eliminate blood, promptly retained in Eppendorf tubes, embedded in liquid nitrogen, and stored at −80°C till the time of real-time PCR (qRT-PCR) analysis for miRNA-137. The other cerebral cortex hemisphere was preserved in 10% neutral buffered formalin to be fixed until the histological investigations and immunohistochemistry test of β-amyloid plaques Aβ1-42.
Cerebral cortex hemisphere tissue levels of MDA and NF-kB were measured using ELISA test reagent kits and ELISA Processing System (The prepared reaction mix samples were applied according to the manufacturing protocol and according to the sandwich technique described earlier.73 StepOne Applied Biosystems, Foster City, USA).
Quantification of miRNA-137 in the hippocampus was determined using the relative gene expression technique of qRT-PCR, which used actin, a housekeeping gene, as an internal reference and the 2Ct method to compute fold changes in the target gene from normal rats as a baseline.74,75 Following the manufacturer’s instructions, the total RNA purification kit extracted the pure RNA from the separated hippocampus after homogenization with a lysis solution. It was done by loading the RNA onto the purification column, which was then eluted with pure RNA under low-ionic strength conditions. The purity was confirmed using UV–Vis-spectrophotometer Q5000 (Quawell Technology, Inc., CA, USA) at OD 260/280 nm and the nanodrop method. The reverse transcription process was used following the manufacturer’s instructions to convert RNA into complementary DNA (cDNA). Specific gene primers, whose sequences are provided in Table 2, were used to build the 2X Maxima SYBR Green/ROX qRT-PCR Master Mix, which was used under the manufacturer’s instructions and to quantify the expression of genes.76
Cerebral cortex hemisphere tissue slides were prepared as indicated by Banchroft and Stevens for histological investigation using conventional hematoxylin and eosin staining.77 In brief, firstly tissues was fixed in a 10% buffered formalin solution to prepared tissues for histopathological evaluation. After ensuring tissue stiffening, tissues were dehydrated in graded ethyl alcohol (50, 70, 95, and 100%) for 2 hours each and then dripped in a paraffin wax three times for 2 hours each time. Paraffin blocks were then used to prepare sections of 4µm thickness cut using the Leica microtome. Finally, sections were deparaffinized and stained properly with hematoxylin and eosin to be examined under a light microscope with the aid of a pathologist.
Tissue amyloid-beta plaques, Aβ1–42, were detected using the Immunohistochemically technique reported previously by Merz et al.78 The procedure involved incubating primary antibodies against amyloid plaques, Aβ1–42 then with secondary antibodies and diaminobenzidine/H2O2. Histopathology has counterstained slide sections with hematoxylin before the slides were inspected under a light microscope.
Statistical analyses of biochemical estimations were performed using one way analysis of variance test followed by Tukey–Kramer multiple comparison test, while statistical analysis of the assigned scores in the histopathological study was performed using the chi-square test using statistical package for social sciences (SPSS; version 19.0) computer software program (SPSS Inc., Chicago, Ill., USA), with the value of p < 0.05 considered statistically significant. Data are expressed as means of 6–10 values ±SEM.
Initially, preliminary screening and critical statistical analysis were investigated to explore the impacts of different variables on the TBLs formulae such as the amount of (lipid, surfactant, bile salt, drug), a hydration medium, and time as well as sonication time. All variables were established to obtain an appropriate size of TBLs with the highest EE%. TBL vesicles were obtained using PL incorporated with cholesterol as lipid phase and SP 60 as a surfactant to obtain higher EE%79,80 and increase the stability of nanovesicles.81 SDC was used as an edge activator at a different amount. As regards the initial survey, the vesicle preparation was perfectly achieved when these conditions involved 50 mg of CIT, 10 mg of cholesterol, chloroform:methanol ratio (2/1) as an organic solvent, and SENS pH 5.5 as a hydration medium. Hydration for 1 h and sonication for 30 min were sufficiently enough for nanovesicles’ formation. The different formulae of the TBLs were prepared following the thin-film hydration process using the various amount of PL, SP60, and SDC and characterized for appropriate physicochemical attributes. In order to investigate the impact of independent variables on dependent ones, the Box–Behnken design was created (Table 3). Regression equations and 3D-response surface plots were utilized to further clarify the link between the dependent and independent variables. In the regression equation, a positive sign denotes a synergistic impact and a negative one an antagonistic one. The 3D-response surface plots exhibit the effect of two independent variables after fixing the third one at the medium-level value.82 The signal-to-noise ratio was calculated with sufficient precision to ensure that the model could be used to explore the design space.83 It is preferable for all dependent parameters (EE percent, VS, ZP, Q8 and Q24h) to have a ratio greater than 4 (Table 1). The adjusted and anticipated R2 values should be comparable to each other in order to have a fair agreement, which was achieved for all parameters (Table 1). The experimental run is listed and calculated along with the measured parameters (Table 3).
Nano platform-based medicine can provide great opportunities to enable drugs to reach their target sites, either passively or actively.84 Table 3 shows the vesicle size values of the formulated CIT-TBLs as the z-average diameter, which represents the mean hydrodynamic diameter of the lipid vesicles.85 The CIT-TBLs were in the nanoscale range, as their VS ranged between 194.8± 7 and 371.7± 25. nm. In Figure 2A, 3D response surface plots are used to graphically depict how PL (x1), SP 60 (x2), and SDC (x3) quantity affect the VS of the TBLs. The size values were subjected to Statistical polynomial analysis and the quadratic model was selected (p<0.0001). Equation 6 shows the quantitative effects of the independent variables on the VS of the TBLs formulae.
(6)
Figure 2 Response surface plot for the effect of PL amount (X1), Sp60 amount (X2) at the middle levels of the third (SDC amount) on (A) vesicle size (B) zeta potential (C) EE%, (D) Q8h, (E) Q24 and (F) desirability of the developed BLs dispersions.
The VS was directly dependent on the effect of PL (x1); as the amount of PL was raised from 100 to 300mg unpredictably, decreasing in the mean VS may be attributable to the availability of more surface area at a high level of PL amount resulting in an accommodating more of CIT into the vesicle bilayer.86,87 The high amount of drug captured in vesicles revealed better packing of the vesicular membrane and a consequent size reduction.88 These findings are similar to those of a previous study.36 As well, the VS was found to significantly increase with increasing Span 60 amount (X2) (p = 0.0012). These findings are in line with previous results, where combining higher content of Span 60 led to higher EE percentage of drug consequently, the whole VS of CIT-TBLS was increased. This may be attributable to the interposition of SP 60 in the bilayer membrane, with the polar head towards the hydrophilic phase.89,90 However, the increase in flexibility and decrease in membrane tension between the aquatic and lipid aspects caused by the reduction in VS and the augmentation of SDC in the nanovesicle lipid bilayer resulted in the creation of smaller droplets at the nanoscale.91 Our findings are consistent with those of Guan et al, who claimed that the smallest ratio of PL: SDC was the main contributor to the production of small particle size of probilosomes containing cyclosporine A.92 Additionally, our findings are consistent with those of Ahad et al, who revealed that a rise in the PL: SDC ratio resulted in a reduction in the particle size of liposomes containing bile salt.93
The stability of CIT-TBLs colloidal dispersions can be partially indicated by zeta potential values which is a crucial characteristic that can influence vesicular properties. Due to electrostatic repulsion, it can dramatically prevent the mutual agglomeration, fusion of nanovesicles and enhances its stability. Abdelbary stated that particles are considered stable when the ZP value is around ±30 mv.94 The ZP of the TBLs formulae ranged between −18.50±8.94 and −38.3±9.56 mV (Table 3).
(7)
Equation 7 shows the quantitative effects of the three independent variables on the ZP of the TBLs formulae. Where X1 is PL, X2 is SP 60, and X3 is SDC. It is found that all the three independent variables had a significant influence on the ZP (p = 0.0003) (Figure 2B). The equation shows that TBLs vesicles with the highest PL amount had the greatest ZP.
Although the PL heads are zwitterions and thus are hypothetically unconvicted at a neuter pH, they nevertheless create a negative ZP value in water due to the orientation of the head group and the formation of water layers on their surface.95 According to Makino et al, charge of a phospholipid bilayer is caused by the orientation of the dipole, which involves the negative charge of the phosphatidyl molecule and the positive charge of the choline molecule in the head group.96
The head group has been shown to be orientated so that the positive choline gathering is inside and the negative phosphatidyl gathering is outside in a medium with low ionic strength, producing a negative surface charge.95 The results demonstrate that the amount of SP60 significantly affected the ZP values of the CIT-TBLs dispersion (P=0.0018). This might be explained by the surfactant-forming vesicles’ lipophilicity. Whenever a surfactant’s lipophilicity is decreased, the ZP values increase because the surfactant’s surface free energy is reduced. This is in harmony with the findings of Abd-Elal et al,97 who investigated the impact of surfactant type and ratio on zolmitriptan-loaded novasomes. They found that when the ratio of this surfactant was increased from 1:1 (SP 60:oleic acid) to 2:1 (SP 60: oleic acid), the ZP of vesicles dramatically improved in vesicular surface charge from (- 63.77±1.12 mV) to (−68.10±10.18 mV). Similar to this, raising the SDC amount dramatically elevated the ZP values. SDC producing negative surface charges and strong electrostatic revulsions between the particles is a feasible explanation.97 These findings point to the vesicles’ strong stability.52 When it comes to the polydispersity index (PDI), a value of 0 represents the ideal and uniform population, whereas a PDI that is almost equivalent to 1 suggests extremely polydispersity vesicles.98 The PDI values in this investigation varied between 0.078 and 0.382, which is indicative of good homogeneity and a restricted particle distribution.99
The prospective role of the formulated TBLs to retain a high amount of CIT is critical for its potential manipulation as a successful brain targeting delivery system. The percent of CIT entrapped within the TBLs ranged from 45.53± 3.35% to 75.35± 3.13% (Table 3). Thus, CIT was successfully entrapped in the CIT-TBLs, indicating that lipid-based TBLs can be used as a delivery system for water-soluble drugs. The increase of PL (x1), SP 60 (x2), and SDC (X3) amount on the EE% of the TBLs are graphically illustrated as 3D response surface plots in Figure 2C.
The ANOVA showed that the best-fitted model for assessing the EE% values was a quadratic model. The resulting equation in terms of coded values shows the quantitative effects of the independent variables on EE% of the TBLs formula as follows:
(8)
It was found that EE% values were significantly affected by all three independent variables (p<0.0002). The regression coefficients indicate that the PL and SP60amount had a noticeable positive effect on EE%, while the SDC amount had negative effects on EE%. The impacts of PL and SP60 amount on EE% at the middle level of the third independent variable (SDC amount) are shown in Figure 2C. Elevating the PL amount from 100 to 300 mg significantly enhanced the EE%(p<0.0001), possibly because there was increased space to incorporate the CIT into TBL vesicles.100 This results in harmony with the study by Singh et al.101 The increase in Span 60 amount leads to significantly higher enhancement in EE. The outcome is consistent with the research by Singh et al.101 An increase in Span 60 results in a noticeably bigger improvement in EE. The increase in the quantity of nanovesicles produced with rising Span 60 concentrations could serve as confirmation.102 The rise in the EE may be related to the large phase transition temperature (50°C) and alkyl chain lengths of Span 60. The drug’s permeability into the bilayer of lipid vesicles is increased by Span 60ʹs long alkyl chain, which raises EE. In direct proportion to the surfactant’s lower HLB value (Span 60, HLB 4.7),103 the drug entrapment and stability would rise.103 These results were consistent with the earlier-reported finding.104
Regarding SDC amount (X3), many studies stated that EE percent of TBLs was decreased as the bile salt concentration increased. They explained this finding as a result of the bile salts’ fluidizing effects on the bilayer membrane of the vesicles, which cause the loading drug to leak out.105 Moreover, a high bile salt concentration causes the development of mixed micelles that increase drug solubility in the hydrous milieu throughout preparation, endangering the EE percent.106,107 In this study, the results are not entirely support these findings, as it is clear that the quantity of SDC increases with a minor decrease in the amount of EE of the drug, and the influence of SDC concentration on EE percent is negligible (p = 0.525).
In vitro release behavior of CIT-TBLs formulae in comparison with that of control (CIT solution in PBS) are demonstrated in Table 3, and graphically profiled in Supplementary Figure 1A and B. The effect of the independent variables on Q8h percentage of the prepared CIT-TBLs formulae was confirmed in 3D-graph (Figure 2D). The data obtained from in vitro release studies proved that the CIT-TBLs could release CIT in a controlled manner. The rate of CIT released from drug solution was significantly higher than the investigated TBLs formulae (p < 0.005). Where drug solution pointed out a 99.99% of CIT release in the first 4 h. In contrast, the prepared TBLs formulae could retard the release of CIT to more than 8 h (42.56 −87.25%). The cumulative percentage of CIT released from TBLs after 8 h (Q8h) was subjected to polynomial analysis and the quadratic model was selected; The ANOVA test for the observed Q8h in CIT-TBLs data indicated that the quadratic model was significant and best fitting for the data (Table 3). The equation of Q8h for CIT-TBLs in terms of coded values is given as follows.
(9)
The equation discloses that the three examined independent variables elucidated a significant impact on the percentage of in vitro CIT release after 8 h (p <0.0001). Due to the amphiphilic characteristics of PL, an increase in the amount of lipid from 100 to 300 mg resulted in a considerable increase in the percentage of drug release after 8 h (p <0.0001).108 The high lipid content followed by low VS, as was previously noted in the VS assessment, therefore an increase in the percentage of drug release is expected. The increasing addition of Span 60 to the TBLs formula was also discovered to be associated with a noticeable rise in the CIT release rate. Indeed, the more association of surfactant particles in the formula provides higher deformability, fluidity, and elasticity for the bilayer membrane of the nanovesicles, which is the favored for drug release.53
Furthermore, there was a favorable correlation between the amount of medication released from TBLS and the increase in SDC amount. The fluidity effect of SDC, which increases lipid bilayer flexibility and facilitates drug leaking from the vesicles, may be responsible for this action.109 Linear kinetic analysis of the CIT release data revealed that most CIT-TBLs formulae were the coefficient of correlation value (R2) best fitted with the Higuchi kinetics equation, as the R2 values predominate over zero-order and first-order kinetics. This indicates the CIT release mechanism by diffusion, as the slow and sustained release of the medication is achieved by its diffusion from the matrix of the TBLs system, as proposed by Higuchi (Supplementary Table 1).
The diffusion studies provide valuable visions about in vivo performance.110 The intranasal permeation analysis was performed on CIT-TBLs formulae and CIT solution, the results obtained are shown in Table 3, and graphically profiled in Supplementary Figure 2A and B. The calculated diffusion parameters for each TBLs formula along with CIT solution are recorded in Table 4. The diffusion parameters including Q24 (μg/cm2), lag time (min), Kp (cm/h), and Enhancement ratios were used to assess the intranasal permeation capability of the tested TBLs formulae against CIT solution. The permeation analysis proved that the developed TBLs formulae exhibited a significantly higher (P<0.05) nasal permeation compared with the CIT solution containing the equivalent amount of CIT. The permeation of the CIT-TBLs formulae ranged from 240.43±12.81 to 538.78± 21.15 μg/cm2 (Table 3), which indicated enhanced permeation compared to the CIT solution (118.30±16.0 μg/cm2).
Table 4 Ex vivo Permeation Parameters of CIT-TBLs Formulae versus CIT Solution
This may have been due to the CIT-TBLs composition involving both phospholipids and edge activators,48,111 their small VS creating a large surface area for enlarged contact of released CIT with the nasal mucosa surface. The intranasal flux (Jss) of the CIT-TBLs formulae ranged from 10.32±0.12 to 22.97±1.83 μg/cm2/h compared to only 5.4±0.69 μg/cm2/h for the CIT solution. The enhancement ratios of the CIT-TBLs formulae compared to the CIT solution ranged from 1.6 to 4.28. Statistical analysis of the Box–Behnken design exhibited that the quadratic model was significant and best fitting for the data. The quantitative impacts of the independent factors on Q24h% of CIT-TBLs are shown in Equation 10 as follows:
(10)
By inspection of the results, the independent variables significantly improved the Q24 of CIT-TBLs (p<0.0001) (Figure 2E). All variables had a positive regression coefficient, indicating their positive impacts on diffusion. ANOVA analysis exhibited that lipid amount (X1) and SP60 (X2) were the higher significant predictors of the quantity of drug permeated after 24 h (p<0.0001).
It can be concrete that increasing lipid amount from 100 to 300mg and SP60 from 20 mg to 50 mg (at a low level of SDC) would increase the quantity of CIT permeated across nasal mucosa after 24 h from 296.12 ± 7 to 538.78 ± 10.35 μg/cm2. Two different attitudes have been proposed for the influenced delivery of medicine by nanovesicles.54,112 The first attitude expects a small vesicle size of CIT-TBLs that resulted in high lipid level and surfactant, which increases the flexibility of the nanovesicles and enables them to pass through the nasal mucosa more easily.54 The bilayer membrane’s extraordinary flexibility allows nanocarriers to squeeze through pores that are much smaller than their sizes.113 Additionally, phospholipids have a strong affinity for cellular membrane, which helps nanovesicles become more permeable. Second, the vesicles serve as drug nanocarriers, sending whole nanovesicles into the nasal mucosa with drug molecules enclosed inside.114 According to the third, nanovesicles can operate as penetration enhancers by allowing nanocarriers bilayers to enter the nasal mucosa and create new pores in paracellular tight junctions, improving the fluidity of the mucosa.115 On the other hand, the permeability coefficient showed a moderately significant improvement after using varied doses (10 mg to 20 mg) of SDC (p < 0.0324). This could be due to the ability of SDC to boost the vesicles’ flexibility by altering the bilayer packing characteristics.52 This would help the vesicles to squeeze easily through the nasal mucosa.116
The optimized TBLs formula was selected by the optimization process of the Box–Behnken design, which generated the requisites of each response variable based on a maximal desirability value of 0.9332 (Figure 2F). This formula was composed of 300mg PL, 43.976mg of SP60, and 20 mg SDC. Optimized TBLs showed a size diameter of 222.94± 3.42 with narrow PDI 0.062±0.013 and zeta potential of −36.37 ± 4.75 mV that provided good stability. Also, optimized TBLs showed EE percentage of 72.92 ± 4.13%, CIT release rate of 85.41 ± 2.07%, and intranasal flux of 528.43 ± 11.65 μg/cm2/h.
The insertion of HA produced a little smaller VS (178±8.92 nm) compared with the un-hyaluronate variants (220±12.67 nm), which is emphasized by the TEM micrograph in Figure 3A and B.
Figure 3 Transmission electron micrograph of the uncoated CIT-TBLs (A) and coated CIT -HA*TBLs (B) of optimized formula.
This phenomenon was explained by Abdelkader et al who stated that the addition of HA reduced the PDI and that increasing the viscosity of the dispersion medium decreased the tendency of nanovesicles to aggregate.117 This lessening in aggregation resulted in the production of TBLs of smaller size than the hyaluronate TBLS. Regarding PDI, the TBLs dispersion is considered mono-dispersed, as the PDI was 0.049±0.012, which was less than 0.062±0.015. In this work, the obtained ZP value of optimized CIT-HA*TBLs was −42±3.6 mV, which is on the negative side due to the immobilization of hyaluronic acid to the nanovesicles’ surface.117,118 Additionally, there was a moderately higher CIT entrapment percentage for CIT-HA*TBLs compared to un-hyaluronate TBLS (74.5% vs 72.92%). This could be linked to hyaluronic acid, the agent that enhances viscosity. As CIT complexation with PL molecules improves, a modest increase in viscosity is expected to result in a rise in EE percent. This follows the results produced by previous literature.40,119
TEM was conducted to investigate the morphology of optimized CIT-TBLs and CIT-HA*TBLs. As observed in Figure 3A and B, the examined sample showed homogeneously, well-identified mono dispersed vesicle with a lipid bilayer without agglomeration. The size of the vesicles measured by Zetasizer was typically following the results obtained using TEM. In addition, a higher magnification demonstrated that the vesicles of CIT-HA*TBLs exhibited a smooth surface and sharp edges, with the concomitance of a HA coat (darker shade) framing the TBLs. This was following the results produced by previous studies.40,119
The dissolution and permeation profiles of CIT from the CIT-TBLs and CIT-HA*TBLs are represented graphically in Supplementary Figure 3A and B. The cumulative release (Q8h percent) and permeation (Q24h μg/cm2) of CIT from CIT-HA*TBLs were diminished compared to unhyaluronated TBLs, as shown in Figure S3A and B (81.27±3.8%, 515.43±12.58% and 85.41±4.13%, 528.43±10.58%, respectively). This slow release in CIT-HA*TBLs could be due to the presence of an HA-coated on the TBLs surface, which increased the thickness of the TBLs layer and leads to the increase of the diffusion distance. Furthermore, CIT-HA*TBLS exhibited high negative ZP, which may result in enhanced TBLs stability and diminished TBLs membrane fusion, hence lowering the CIT release out of TBLS.
The above results suggest that CIT-HA*TBLs would be stable in the blood circulation and would be released at a slow rate at the target site.118,120
DSC thermograms of the pure drug (CIT), excipients (PL, SP 60, SDC, CHO, HA, and CKC) drug –excipients mixture (physical mix in a ratio of 1:1), and the optimized CIT-HA*TBLs formula are shown in Figure 4. The DSC thermograms of CIT exhibited one endothermic peaks at 270 0.69°C, as reported previously for CIT121 (Figure 4A). Concerning PL 90 G, the thermal analysis shows three endothermic peaks at 232.21°C, 335.03 °C, and 365.48°C (Figure 4B). SP60 thermograms shows one endothermic peak at 56.18°C (Figure 4C).
Figure 4 DSC thermograms of CIT, PL90 G, SP60, SDC, HA, CKC, CHO, physical mixture and CIT-HA*TBLs-coated nanovesicles formula.
The thermograph of SDC showed two endothermic peaks at 87.20°C and 236.45°C and two an exothermic recrystallization peaks at 195.02°C and 335.22°C (Figure 4D).122 CHO thermal analysis displays one endothermic peak at 143.94°C and another exothermic peak at 352.94°C (Figure 4E). The thermograms of HA exhibited an endothermic peak at 82.61.94°C and another two exothermic peaks at 238.28°C and a peak at 391.98 (Figure 4F). Concerning CKC, the thermal analysis shows one broad endothermic peak started at 44.71°C and 195.84 (Figure 4H). The thermograms of (the physical Mixture and optimized CIT-HA*TBLs formula) indicated CIT melting endothermal and exothermal subsidence (Figure 4G–I). The CIT’s distinctive exothermic peak had disappeared, and the endothermic peaks of the surfactant/lipid bilayers had widened and/or altered. This may explain the increased trapping of CIT in the HA*TBLS in an amorphous state. It also suggests that CIT interacts with the bilayer components, generating many lattice defects.
The optimized CIT-HA*TBLs formula’s stability studies were assessed for EE percent, Zp and VS after 30, 60, and 90 days at 4°C as shown in Figure 5. The formula of CIT-HA*TBLs showed minor variations in EE percent (reduced from 74.55 to 71.69 4.83) and PS (enlarged from 178.8 to 183.5 nm). Zp changed throughout the stability testing, going from −42 2.65 mv to −40 1.87 mv. A one-way ANOVA test revealed these small changes to be unimportant (P > 0.05).
Figure 5 Effect of storage at 4℃ for 3 months on entrapment efficiency (EE), vesicle size (VS) and Zeta potential of the optimized CIT-HA*TBLs formula.
According to earlier theories,52 the high rigidity of the lipid membrane of the nanovesicles and their capacity to hold the medication may be the causes of the optimized formula’s physical stability.
Incorporating the CIT-HA*TBLs formula with the poloxamer 407, poloxamer 188, and CAP971 polymers results in the proper viscosity and bioadhesive properties.123 The hyaluronated CIT-HA*TBLs formula and the thermogel bases were successfully combined. At body temperature and room temperature, the viscosities of the CIT-HA*TBLs thermogel were 8604.96±43.4 and 648.37±35.7 cp, respectively. Good pseudo-plasticity and thixotropic behavior were confirmed by rheological parameters with Farrow’s constant (N) values higher than 1.
The hysteresis loop area gave values of 2446.7and 1839.4 dyne/cm². sec for the thermogel at room and body temperatures, respectively. Comparing the release and ex vivo permeation patterns of the CIT-HA*TBLs thermogel to the Control thermogel is shown in Figure 6A and B. The findings demonstrate that the CIT-HA*TBLs thermogel performed better than the control CIT thermogel in terms of low release and high Q24. Citicoline was released from the CIT-HA*TBLs thermogel formula at a rate of 63.82.75%, compared to 77.571.62% for control thermogel over an 8-hour period. This is illustrated by the fact that the TBLs nanovesicles capture the CIT medication, which prolongs and sustains the CIT release compared to the un entrapped drug. Additionally, the CIT-HA*TBLs showed significantly more drug permeation than the control thermogel after 24 hours 443.1±3.3 μg/cm²).
Figure 6 Dissolution profiles (a) and permeation profiles (b) of CIT from the control thermogel and CIT-HA*TBLs hermogel.
The CIT-TBLs *HA thermogel’s gelation temperature provided a good value of 33.095 that is thought to be suitable for nasal application. This degree causes it to be in a fluid state at room temperature, making administration simpler and dosage calculations more accurate. It also causes it to become an adhesive gel with increasing residence duration near the lower limit of the nasal physiological temperature range (32–37c).
The mean value results of rats subjected to AlCl3 significantly raised hippocampal tissue homogenate levels of NFκB and MDA to 6.83± 0.37 and 5.07± 0.10 by 421.60% and 603.57%, respectively, as compared to the normal control group 1.62 ± 0.12 and 0.84 ± 0.02. On the other hand, treatment with CIT solution, Control thermogel, CIT-TBLs thermogel, CIT-HA*TBLs thermogel, and HA*TBLs thermogel via an intranasal application for consecutive 2 weeks restoring both NFκB 6.06± 0.08, 5.08± 0.05, 4.42 ± 0.27, 2.00± 0.00 and 4.38± 0.33, respectively, by 88.73%, 74.48%, 64.72%, 29.28%, and 64.13% as compared to positive control rats. Additionally, MDA mean results represent significant decrease when compared to positive control group to 4.13 ± 0.12, 3.66± 0.08, 2.95 ± 0.10, 0.99± 0.01, and 3.20± 0.05 by about 81.48%, 72.29%, 58.18%, 19.53 and 63.12%, respectively. The intranasal applied CIT-HA*TBLs thermogel restored both NFκB and MDA to normal levels (Table 5). Our observed effect represents CIT-HA*TBLs thermogel protecting animals from aluminum chloride-induced neurodegenerative damage. Previous data represent that AlCl3-induced memory impairment is thought to be caused by a variety of ways124 AlCl3 cognitive toxicities have been linked to the upregulation of APP, Ab, NF-kB, AChE activity, nitric oxide release, oxidative stress, and the eventual neuronal apoptosis.125,126
Table 5 Presentation of Cerebral Cortex Hemisphere Tissue NFκB and MDA Concerning the Effect of 2 Weeks (0.9mg/kg) Intranasal Administration of Treatment in Different Experimental Groups
Furthermore, Abdel-Zaher et al127 indicated aluminum poisoning damages cytoplasmic membranes and disrupts neuronal integrity, whereas citicoline protects neurons from this damage and improves memory. Consequently, d-galactose-induced subacute aging models reveal memory loss and neuroinflammation linked to NFκB activation/reticuloendotheliosis viral oncogene homolog A (RelA) activation and decreased sirtuin 1 (SIRT1) expression in the hippocampus. Salidroside reduces memory impairments and inflammatory mediators, including TNF-and IL-1 in d-gal-treated animals. SIRT1 is up-regulated by salidroside, inhibiting the NFκB pathway.128,129 Otherwise, the lipopolysaccharide mice sepsis model reported that the NFκB transcription factor regulates the generation of pro-inflammatory factors via the phosphatidylinositol‐3‐kinase/protein kinase B (PI3K/Akt) signaling pathway.130
In addition, the streptozotocin-induced neurodegenerative disorder mice model indicates that liposomal citicoline therapy increases pro-inflammatory cytokines NF-κB, TNF-, IL-6, IL-8, and MCP-1.131,132 All these data confirmed our results that intranasal citicoline significantly reduces NFκB hippocampus tissue levels (Table 5). In this study, the administration of AlCl3 resulted in learning and memory impairments, and an increase followed this in oxidative stress levels; an increase in hippocampus MDA levels. According to several previous studies, aluminum chloride can cause a cytotoxic effect via the induction of oxidative stress.133 Indeed, the AlCl3-induced parkinsonism model depended on apoptosis, and oxidative stress biomarkers increased, especially MDA and Catalase (CAT).134 Also, according to Henderson et a.135 Reactive oxygen species (ROS) production causes neuronal cell death and apoptosis via Bax expression and mitochondrial translocation.
Furthermore, the glial fibrillary acidic protein (GFAP) upregulation in astrocytes has been linked to Aβ1-42 and oxidative stress produced by amyloidogenic-hyperlipidemic and other pathogenic processes of AD.136 Additionally, oxidative stress activates nicotine amide adenine dinucleotide phosphate (NADPH) oxidase, which induces the production of neuroinflammatory markers, including tumor necrosis factor-alpha TNF-α and NFκB, which contribute to AD development.137,138 In addition to amyloid plaque buildup, AD transgenic animal’s exhibit oxidative brain injury.139 Consequently, stress-activated protein kinases, including c-Jun N-terminal kinases (JNK), p38 mitogen-activated protein kinases (MAPKs), and extracellular-signal-regulated kinases 1/2 (ERK1/2), and changes in redox-sensitive transcription factors may have a role in Alzheimer’s disease pathogenesis, according to the findings of these studies.140–142 Most previous studies on oxidative damage in AD have explained why the AD brain produces more ROS.143 Mitochondrial malfunction due to oxidative stress may also play a role in developing ROS, particularly with the metal liganded with Aβ.144
In agreement with Al-Kuraishy et al,145 citicoline increased psychomotor vigilance, arousal, and visual working memory while reducing oxidative stress in healthy participants. Also, the research found that giving adolescent males CIT for consecutive 28 days improved psychomotor function, cognitive-attention activity, and reduced impulsivity146 Recently, neuroprotection has been focusing on the role of inflammation and oxidative stress.147
Furthermore, citicoline has been shown to protect against neurodegenerative disorders in animal and human trials.148 All these data agree with our results that the intranasal CIT has been modulating MDA brain tissue levels (Table 5).
The miRNA-137 regulates glutamatergic synaptic transmission and plasticity in the hippocampus by modulating genes’ translation at different ends of the synapse. The current functional miRNA-137 data support the idea that miRNAs can modulate complex gene regulation networks implicated in common physiological pathways. From this point of view, our mean value data represent that miRNA 137 genetic expression significantly increased via aluminum chloride exposure to 7.01± 1.32 by 701% compared to the normal control rat’s mean of 1.00 ± 0.01.
In contrast, treatment with CIT Solution, Control thermogel, CIT-BLs thermogel, CIT-HA*TBLs thermogel, and HA*TBLs thermogel via an intranasal application for consecutive 2 weeks represent a significant decrease in miRNA 137 mean gene expression values to 5.09± 0.53, 4.19± 0.14, 3.43± 0.52, 1.76± 0.21 and 3.88± 0.31, respectively. The CIT-HA*TBLs thermogel restoring miRNA 137 mean genetic expression back to normal compared to the positive control group and the normal one (Figure 7). All recent studies have shown that miRNA is involved in neurodegenerative disorder, although the initial patient investigations on synapse represent elevated miRNA-137 levels.149 Early glutamatergic synaptic formation and aberrant glutamatergic synaptic maturation have recently been established either in AD or schizophrenia patients. Both are likely results of lost miRNA-137 function during brain development in severe cognitive impairments.150,151
Figure 7 Bar chart presentation of hippocampal tissue miRNA 137 gene expression levels concerning the effect of 2 weeks (0.9mg/kg) intranasal administration of CIT Solution, Control thermogel, CIT-TBLs thermogel, CIT-HA*TBLs thermogel, and HA*BLs thermogel against AlCl3-induced AD disease. AD: Alzheimer’s disease. The mean of 6–8 values ± standard error of the mean (S.E.M). aSignificantly different from the normal control group, bSignificantly different from the positive control group, at p < 0.05.
Additionally, the postmortem prefrontal cortex tissue revealed that single nucleotide polymorphism homozygote was related to reduced miRNA-137 expression, which may induce bipolar or schizophrenia152,153 Moreover, a kainic acid promotes hippocampus seizures convulsion in mice, indicating that Interhippocampal injection of anti-microRNA oligonucleotides targeting microRNA-124 and −137 normalized neuroblast proliferation and prevented memory loss and nerve damage154 Furthermore, the latest research suggests that miRNA-137 may have a role in drug addiction inpatient for methamphetamine cessation155 although its exact involvement remains uncertain.
The microRNA-137 affects neuronal growth and synaptic plasticity156 Recently, in an in vitro Alzheimer’s disease model, the SNHG19/hsa-miRNA-137 epigenetic axis regulates Aβ 25–35-induced human neuroblastoma cell line (SH-SY5Y) cytotoxicity.157 Otherwise, the prostate cancer experimental model indicates that deregulation of microRNA significantly regulates MDA levels and inhibits cell proliferation, metastasis, and apoptosis.158 The modulation of microRNA circulating levels has also been shown to regulate ROS levels and trigger cell autophagy in a breast cancer model, which is beneficial to patient therapy159 Additionally, the Kruppel-like factor 15 (KLF15) is downregulated by miRNA-137-3p overexpression in an ischemia/reperfusion-induced cardiomyocyte animal model study.160 Indeed, all recent studies confirmed our data results from miRNA-137-3p downregulation’s critical role in cell survival, preventing cell apoptosis and neural damage.
The microscopic examination of brain sections from the normal group revealed normal cerebral cortex and different hippocampus regions. On the contrary, the positive control group showed diffuse gliosis with thickened and congested blood vessels in the cerebral cortex. Numerous dark degenerating neurons were detected. Group 3 received CIT-Solution and showed the variable number of dark degenerated neurons in the cerebral cortex associated with mild-to-moderate vascular thickening, fewer degenerated neurons were detected in the CA-2 and CA-4 regions of the hippocampus. Brain sections from Group 4 received Control thermogel showed degenerating neurons in the cerebral cortex with neuronophagia either some of the examined sections showed proliferating cerebral vasculature coupled with hippocampus exhibited few degenerating neurons in its different regions.
In contrast, Group 5 received CIT-TBLs thermogel normal cerebral cortex examination coupled with a hippocampus few dark degenerating neurons scattered in the different regions. Concerning the brain section from Group 6 CIT-HA*TBLs thermogel represent cerebral cortex and hippocampus regions of the examined brain sections were apparently regular. While Group 7 received HA*TBLs thermogel showed apparently normal cerebral cortex in some of the examined sections with neuronal degeneration and neuronophagia in some other sections. Hippocampus showed variable number of shrunk degenerating neurons scattered in different regions (Figure 8).
Figure 8 Microscopic photomicrograph brain H&E staining with a higher magnification represents; (1A–1E) Normal control sections (25 µm) showing the normal histological structure of cornu ammonis hippocampus pyramidal neurons (CA-1- CA-2, CA-3, CA-4) and dentate gyrus (DG); (2A–2E) Positive control group showing numerous dark degenerated neurons in CA-1- CA-2, CA-3, CA-4 and DG regions (black arrow); Group 3 received CIT drug solution (3A) showing some degenerating neurons in the cerebral cortex (black arrows) with mild vascular thickening (red arrow), (3B and 3C) sections showing few degenerating neurons (black arrows) in CA-1, CA-2, CA-3, (3D) sections showing few degenerating neurons CA-4; (3E) showing some degenerating neurons with DG region of hippocampus; sections from Group 4 received Control thermogel (4A) showing neuronal degeneration and neuronophagia (black arrows) in cerebral cortex with congested blood vessels, (4B–4D) showing some degenerating neurons (black arrows) in CA-1 CA-2, CA-3, CA-4 region of hippocampus and 4E: showing some degenerating neurons in DG region of hippocampus; Group 5 received CIT-TBLs thermogel 5A: showing apparently normal cerebral cortex, (5B and 5C) showing few degenerating neurons in CA-1, CA-2, and CA-3 region of hippocampus while (5D) showing CA-4 hippocampus region apparently normal structure and (5E) showing few degenerating neurons in DG region of hippocampus; Group 6 received CIT-HA*TBLs thermogel (6A) showing apparently normal cerebral cortex, (6B) showed average normal hippocampus and (6C) represent a normal cerebral cortex and (6D) showing apparently normal CA1 region of hippocampus (black arrows) normal histological structure of DG regions; Group 7 receiving HA*TBL thermogel (7A) showing few neuronal degenerations with neuronophagia (black arrows) in the cerebral cortex; (7B–7D) showing number of degenerating neurons in the CA-1, CA-2, and CA-3 region of hippocampus (black arrows) and CA4 showing number of degenerating neurons in the region of hippocampus with marked cellular loss (black arrows); (7E) showing number of degenerating neurons in the DG region of hippocampus.
Additionally, the severity of histopathological alterations in different experimental group’s brain regions, cerebral cortex, and hippocampus was evaluated by assigning semi-quantitative score quantitative analysis, with a score of – assigned the absence of any detectable lesions in normal brain sections, + sections with mild histopathological changes, ++ assigned to the sections that represent moderate histopathological changes, while a severe represented lesion was assigned with +++ label CIT-TBLs thermogel, CIT-HA*TBLs thermogel, and HA*TBLs thermogel sections showed significantly lower scoring compared with positive control AD sections with restoring vascular brain integrity to normal (Table 6). Almuhayawi et al,160
Table 6 Histopathological Lesion Score in Cerebral Cortex and Hippocampus Regions in Different Experimental Groups
Alzheimer’s rat model represents induction of AD-induced unorganized cerebral cortex with shrinkage neuron treated. Citicoline’s crucial significance in reestablishing normal brain and cerebral cortex structure is also demonstrated by tramadol-induced neurodegenerative diseases.161 Furthermore, the epilepsy rat model treated via the citicoline intranasal route potentially enhances neurons.162 These data supported our histopathological examination of the brain and hippocampus region (Figure 8).
Negative immune staining for Aβ1-42 was detected in both the cerebral cortex and hippocampus of the normal control group. On the contrary, the positive control group showed increased immune reactivity expression of Aβ1-42 in both the cerebral cortex and hippocampus that increased to about 42.1-fold compared to the Normal group. Brain sections of Group 3 received CIT solution revealed moderate positive immune staining expression for Aβ1-42 decreased to about 18.3-fold compared to positive control group. Likewise, Group 4 received Control thermogel exhibited moderate amounts of Aβ1-42 immune staining expression in the examined brain sections to about 20.2-fold compared to the positive control group. Group 5 received CIT-TBLs thermogel all examined brain sections noticed a minimal negative Aβ1-42 immune staining that decreased to about 31.1-fold compared to the positive control group.
Additionally, negative expression of Aβ1-42 detected in all examined sections of Group 6 received CIT-HA*TBLs thermogel that decreased to about 60.9-fold and minimal Aβ1-42 expression for Group 7 received HA*BL thermogel decreased to about 46.8-fold compared to the positive control group (Figure 9A and B). Our results indicated that the application of intranasal CIT-HA*TBLs thermogel for 2 weeks restored brain and extracellular matrix structure via suppressing Aβ1-42 expression.
Figure 9 (A) Photomicrograph of brain Aβ1-42 immune staining showing at higher magnification 15 µm and 25 µm for Group 1 normal control group negative reactivity to Aβ1-42 staining (Figure 1A and B); The positive control group showing strong reactivity for Aβ1-42 staining (Figure 2A and B); Group 3 received CIT solution, and Group 4 received control thermogel showing moderate reactivity for Aβ1-42 staining (Figures 3A, 3B; 4A and 4B); also Group 5 received CIT-TBLs thermogel represents a minimal negative reactivity toward Aβ1-42 staining (Figure 5A and B); Group 6 CIT-HA*TBLs thermogel showing negative reactivity toward Aβ1-42 staining (Figure 6A and B); Group 7 received HA*TBL thermogel showing minimal positive reactivity staining for Aβ1-42 staining (Figure 7A and B). (B) Statistical analysis of Aβ area expression was conducted in different experimental groups. Data is presented as means ±SE, significance was considered at P˂0.05.], *Significantly different from control at P < 0.05, ** Significantly different from positive control at P < 0.05.***Significantly different from CIT solution at P < 0.05. ****Significantly different from CIT solution at P < 0.05. ns No Significantly different from presented at P < 0.05.
In agreement with our results, a high-fat diet/overectomy/d-galactosamine-induced Alzheimer’s disease rat model confirmed the elevation of Aβ1-42 expression in the diseased animal that was down-regulated by perindopril treatment in the brain hippocampus.163 Also, many researchers enrolled that the imbalance of homeostasis for Ach resulted in the deposition of Aβ-induced neural damage; indeed, the drug treatment enhancing Ach levels promotes Aβ degradation restoring neural damage caused via Alzheimer’s disease induction.164–166
In the current study, a brand-new nano platform carrier (TBLs) for the intranasal administration of CIT for the treatment of AD was created. TBLs were created using the method of thin-film hydration and were appropriately optimized using the Box–Behnken design. The improved TBLs composition, which included 300 mg of PL 9o G, 43.97 mg of SP 60, and 20 mg of SDC, was decorated on the surface with HA. The high EE percentage of 74±45, sustained-release Q8h of 81.27±5.4%, high permeation Q24h of 512.43±19.58 µg/cm2, minor VS (178±8.92 nm), and high ZP (42±3.6 mV) were assessed for the CIT-HA*TBLs formula. The CIT-HA*TBLs thermogel was also able to overcome the two main barriers to CIT’s passage through the BBB, which are its high hydrophilicity and rapid metabolism. The CIT was professionally and quickly delivered to the brain via the intranasal route owing to the designed CIT-HA*TBLs thermogel. The CIT-HA*TBLs thermogel significantly outperformed in pharmacodynamic tests than the CIT-TBLs thermogel, CIT-thermogel, HA-TBLs thermogel, and CIT solution. According to the in vivo findings, we came to the conclusion that CIT-HA*TBls formula might be promising agents for treating neurodegenerative disorder by reversing hippocampus neurodegenerative injury by boosting cholinergic activity through down-regulation of hippocampus miR-137 and Aβ1–42 expression in conjunction with a reduction in oxidative stress biomarker release MDA and inflammatory NF-kB biomarkers. The CIT-HA*BLs thermogel offers a potential formula for restoring the integrity of the hippocampus during neurodegenerative disorders, which permits it to be employed as a neuroprotective and anti-. Alzheimer’s drug. This is strongly supported by the results of this research.
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