Anti-depressant effect of Naringenin-loaded hybridized nanoparticles in diabetic rats via PPARγ/NLRP3 pathway

Naringenin (NAR) has various biological activities but low bioavailability. The current study examines the effect of Naringenin-loaded hybridized nanoparticles (NAR-HNPs) and NAR on depression induced by streptozotocin (STZ) in rats. NAR-HNPs formula with the highest in vitro NAR released profile, lowest polydispersity index value (0.21 ± 0.02), highest entrapment efficiency (98.7 ± 2.01%), as well as an acceptable particle size and zeta potential of 415.2 ± 9.54 nm and 52.8 ± 1.04 mV, respectively, was considered the optimum formulation. It was characterized by differential scanning calorimetry, examined using a transmission electron microscope, and a stability study was conducted at different temperatures to monitor its stability efficiency showing that NAR-HNP formulation maintains stability at 4 °C. The selected formulation was subjected to an acute toxicological test, a pharmacokinetic analysis, and a Diabetes mellitus (DM) experimental model. STZ (50 mg/kg) given as a single i.p. rendered rats diabetic. Diabetic rat groups were allocated into 4 groups: one group received no treatment, while the remaining three received oral doses of unloaded HNPs, NAR (50 mg/kg), NAR-HNPs (50 mg/kg) and NAR (50 mg/kg) + peroxisome proliferator-activated receptor-γ (PPAR-γ) antagonist, GW9662 (1mg/kg, i.p.) for three weeks. Additional four non-diabetic rat groups received: distilled water (normal), free NAR, and NAR-HNPs, respectively for three weeks. NAR and NAR-HNPs reduced immobility time in forced swimming test and serum blood glucose while increasing serum insulin level. They also reduced cortical and hippocampal 5-hydroxyindoeacetic acid, 3,4-Dihydroxy-phenylacetic acid, malondialdehyde, NLR family pyrin domain containing-3 (NLRP3) and interleukin-1beta content while raised serotonin, nor-epinephrine, dopamine and glutathione level. PPAR-γ gene expression was elevated too. So, NAR and NAR-HNPs reduced DM-induced depression by influencing brain neurotransmitters and exhibiting anti-oxidant and anti-inflammatory effects through the activation PPAR-γ/ NLRP3 pathway. NAR-HNPs showed the best pharmacokinetic and therapeutic results.


Preparation of hybridized nanoparticles
The previously reported self-assembly method [38][39][40] was used for the preparation of NAR-HNPs with modifications.Briefly, 50 mg NAR dispersed in aqueous solution of 0.25% w/v poloxamer F12750 mg was added dropwise under magnetic stirring to 1% v/v aqueous acetic acid solution containing CS (0.2 or 0.4% w/v).The lipid matrix was prepared as 2% w/v or 4% w/v GMO with or without 2% w/v lipoid S45 dissolved in 5 ml ethanol.The drug polymer solution was gradually supplemented with the lipid matrix.The resultant mixture was magnetically stirred for 30 min at room temperature.After that, the mixture was placed in a water bath at room temperature and subjected to a probe-type ultrasonicator treatment for 5 min at a power level of 150W to create NAR-loaded

Entrapment efficiency measurement (EE%)
The entrapment efficiencies of the formulated nanoparticles were calculated indirectly through the separation of the unentrapped NAR from the one that was trapped in the NAR-HNPs by centrifugation at 10,000 rpm for 60 min at 4° (Union 32R, Hanil, Korea).The supernatant was collected, passed through a 0.2-micron Millipore membrane filter, and then suitably diluted with methanol before being spectrophotometrically analyzed.at 289 nm (Shimadzu UV spectrophotometer, 2401/PC, Japan) 42,43 .The entrapment efficiency was calculated using the following equation:

Particle size analysis (PS)
The particle size measurement was conducted after tenfold appropriate dilution of the NAR-HNPs with distilled water using a Zetasizer (Malvern Instrument, Worcestershire, UK) at a fixed angle of 90° at 25 °C.Each value was the average of three measurements.

Zeta potential measurement
The particle charge was measured using a Zetasizer at 25 °C.Each sample was required to be sufficiently diluted tenfold with demineralized particle-free water before measurement.

In vitro release of NAR from the hybridized nanoparticles
The dialysis bag diffusion technique was used to compare the in vitro release of NAR from NAR-HNPs to the free NAR 44 .The sample was put in the dialysis membrane, sealed at both ends, and then submerged in 100ml of release media (phosphate buffer pH 6.8) that was stirred at 50 rpm and kept at 37 ± 0.5 °C45 . 2 ml of the sample was taken out of the release medium and replaced with the same volume of the fresh release medium at the predetermined time intervals (0.5, 1, 2, 3, 4, 5, 6, 7, 8 and 24 h).At 322.8 nm, the amount of NAR released was quantified spectrophotometrically.The experiment was done in triplicate, and the results were presented as mean values with standard deviations.A graph showing the cumulative percentage of NAR released over time was illustrated.

Characterization of the selected NAR-HNPs
Regarding the results obtained from the entrapment efficiency (EE), particle size (PS) analysis, zeta potential (ZP) measurement and the in vitro release of NAR from the prepared HNPs, the formulation showing the highest EE%, acceptable PS and maximum percent of NAR released after 24 h was chosen for further characterization and for the in vivo evaluation.

Differential scanning calorimetry (DSC)
The thermal characteristics of NAR, CS, lipoid S45, and the optimized NAR-HNPs were determined using DSC (Shimadzu DSC-60 apparatus, Shimadzu Corporation, Kyoto).About 5 mg sample was weighed accurately into standard aluminum pans using an empty pan as a reference.Under liquid nitrogen (25 ml/min), the samples were heated from 30 to 300 °C at 10 °C per minute.The thermograms generated were documented and the effects of NAR inclusion in the nanoparticles were assessed.

Morphology examination of NAR-HNPs
Transmission electron microscopy (JEOL, JEM-1230, Tokyo, Japan) was used to examine the morphology of the optimized NAR-HNPs.One drop of the diluted sample was stained with 2% (w/v) phosphotungestic acid for 30 seconds at room temperature, and then it was deposited on carbon-coated grids with films for analysis.

pH measurement
The pH of selected formulation was determined at a temperature of 25 °C using a pH meter (Jenway, Bibby Scientific Limited, Staffordshire, UK).The pH meter was originally standardized using buffer solutions with pH values of 7.0 and 10.0.0.5 gm of NAR-HNPs had been completely mixed and diluted ten times with bi-distilled water, pH was determined.Three measurements, including the mean and standard deviation, produced the values that were displayed.

Study of NAR-loaded HNPs stability
The impact of one month storage on both physical and chemical properties was examined 46,47 , to test the stability of the optimized formulation.NAR-HNPs was kept in screw-capped vials in the dark and stored at 4 °C, 25 °C, and 40 °C48 .Every 10 days, samples were taken, evaluated for EE%, PS, ZP, and PDI, and then compared to freshly made formulation.Three duplicates of each experiment were carried out.

Linearity
The acceptable range for linearity in analytical chemistry, particularly for calibration curves in methods like HPLC, The R2 value should generally be close to 1.0 (preferably greater than 0.99).It represents the proportion of the variance in the dependent variable (response) that is predictable from the independent variable (concentration).

Precision and accuracy
The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) provides guidelines for the pharmaceutical industry, including guidance on validation of analytical procedures.Acceptance criteria for precision are typically defined in terms of relative standard deviation (RSD) or percentage relative standard deviation (%RSD) for peak area.However, specific %RSD values ranged between 2 and 5%.
In addition, in HPLC, accuracy is a fundamental parameter that ensures the measured results from the method are close to the true or accepted values for the analyzed analytes.The accuracy limit in HPLC is typically expressed as a percentage relative error or percentage recovery.It's important to achieve accurate results to ensure the reliability and validity of the analytical data obtained.The acceptable accuracy limit in HPLC is often defined within a specific range, commonly between 98 and 102%.

Pharmacokinetic study of NAR
Rats (200 and 230 g) were randomly divided into 2 groups, 6 rats each.Rats were food-fasted for 12 h.Rats in group 1 were orally administered free NAR (50 mg/kg) suspended in sodium carboxymethylcellulose (0.5%).Group 2 rats were orally administered an equivalent dose of NAR-HNPs (50 mg NAR dispersed in aqueous solution of 0.25% w/v poloxamer F12750 mg was added dropwise under magnetic stirring to 1% v/v aqueous acetic acid solution containing CS (0.2 or 0.4% w/v).Following drug administration, blood samples (about 0.5 ml) were drawn at 0.25, 0.5, 1, 2, 3, 4, 6, 8, 24, and 48 h.Blood samples were centrifuged right away for 10 min; separated plasma were aliquoted and kept at − 80ºC until analysis.Dose of NAR was chosen in relevance to previous study 50 .

Preparation of plasma samples
Samples were extracted twice with ethyl acetate, stirred for three minutes, and centrifuged for five minutes at 3,500 rpm.The combined supernatants were then dried and reconstituted with acetonitrile, and 20 μl of the reconstituted supernatant was injected into HPLC for quantitation.

Instrumentation and chromatographic conditions
The Agilent HPLC system (HP 1100 series), which includes a quaternary pump, on-line degasser, auto-sampler, and UV detector, was used to analyse the plasma samples under the control of the Chemstation software (Hewlett Packard, Waldbronn, Germany).Agilent Zorbax XDB C18 column (150 × 4.6 mm I.D., 5 m, Germany) served as the analytical column, and a guard cartridge containing a comparable stationary phase served as the guard.NAR was isocratically eluted at pH 2.5 and 1.0 ml/min using acetonitrile, water, and formic acid (21:78.8:0.2).The 280 nm detection wavelength.By comparing the peak areas of the sample to those of standard solutions of NAR, the NAR concentrations in the plasma samples were calculated 51 .

Pharmacokinetics and statistical analysis
The pharmacokinetic parameters for NAR or NAR-HNPs were derived using the PK Solver 2.0 'add-in' for Excel 2010 software.The data were presented as mean ± SEM.GraphPad Prism 8.0 for Windows was employed for all statistical analyses (GraphPad, San Diego, CA, USA).

Body weight change
On both the initial and final days of the experiment, each animal's body weight was measured, and the following formula was used to figure out the percentage change in body weight:

Behavioral analysis
Activity cage test A grid floor activity cage (Model No. 7430, Ugo-Basile, Comerio, Italy) was employed to assess locomotor activity.The rat's movements that disrupted infrared beams were automatically tracked, and the apparatus software used this information to count the number of times the rat moved horizontally.Before administering STZ, rats were acclimatized to the test room for 1 h.Then, each rat was put in the activity cage for 5 min, and the basal activity counts were written down.When the test is over, each rat was removed carefully from the apparatus and returned to its home cage.To prevent odor cues, the arena was cleaned with a 70 percent (v/v) alcohol solution in distilled water after each rat.Each rat was tested again for 5 min with the activity apparatus 24 h after the drug was given, and the final activity counts were written down 56

Forced swimming test
A vertical cylinder made of plexiglass measuring 46 cm in height and 20 cm in width was filled with tap water heated to between 23 and 25 °C to a depth of 30 cm.After 23 days of STZ injection, the FST was performed to the animals.Each rat was placed in the Plexiglas cylinder containing water for two swimming sessions.During the pre-test, the rat was required to swim for 10 min.Following the pre-test, the rat was withdrawn from the cylinder, dried, and returned to its home cage.After each test, the water in the cylinder was refilled.The rat was re-subjected to the same experimental settings for 5 min 24 h later.A camera above the cylinder filmed the test so that it could be studied later.The rat was immobile when it ceased moving and floated in the water with its head just above the surface, without moving 57 .

Blood glucose level
A portable glucometer (OKmeter, OK Biotech) was used to assess blood glucose levels 24 h after the last treatment.A drop of blood was taken from each rat using the tail vein puncture technique and placed on the glucometer strip loaded in the device for blood glucose measurement.

Sample preparation
Rats were decapitated after behavioral testing.Cortical and hippocampal brain tissues were extracted, immediately weighed to prevent any effects from drying, and stored at 80 °C after a meticulous brain removal.Other brain samples were used for quantitative real-time PCR analysis.The homogenizer MPW-120 from medical instruments homogenized cortical and hippocampal brain tissues in 10% (w/v) ice-cold phosphate buffer.The homogenate was centrifuged using a cooling centrifuge (2k15; Sigma/ Laborzentrifugen) at 4000 rpm for 5-20 min.The collected supernatant was then used to measure biochemical analysis.

Gene expression of PPARγ
According to the manufacturer's guidelines, total RNA was isolated from brain (hippocampus and cortex) tissue with GENEzol™ TriRNA Pure kit (Geneaid®).The level and purity of RNA were determined with Nanodrop spectrophotometer.RevertAid-first sreand cDNA synthesis kit (Thermo Fisher®invitrogen ™) was used to synthesize cDNA from total RNA(2.0µg)according to manufacturer's guidelines.

Determination of monoamines content
The concentrations of cortical and hippocampal monoamines (NE, 5-HT, DA) as well as 5-Hydroxyindoleacetic acid (5-HTAA), and 3,4-Dihydroxyphenylacetic acid (DOPAC), metabolites in micrograms per gram of brain tissue were determined using HPLC (Agilent 1200 series; Agilent Technologies, California, USA) as previously described before 61 , and were calculated as follows: Where AT is the area under the curve for the sample, AS is the area under the curve for the standard, CS is the standard concentration (μg/ml), and the dilution factor is 10.

Statistical analysis
Results are displayed as mean and SEM.Except for the activity cage test and stability study, in which two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test was used for analyses of activity cage test and two-way ANOVA followed by Bonferroni multiple comparison test was used for analyses of the stability study, all other analyses were conducted using one-way ANOVA followed by LSD test.Windows GraphPad Prism 8.0 was used for all studies (GraphPad, San Diego, CA, USA).The cutoff for statistical significance was p < 0.05.

Results
Preparation and characterization of NAR-HNPs NAR-loaded HNPs were successfully prepared using the self-assembly method, and the detailed compositions are presented in Table 1.As evidenced by the nanoparticle's composition, poloxamer F127, which has been proven to have a high affinity for GMO in creating liquid crystal nanoparticles, was utilized as a stabilizer to help incorporate GMO into the nanoparticles 39 .Moreover, the presence and absence of lipoid S45 during the NAR-HNPs preparation were studied.The results of the measurements for the EE%, PS, ZP, and polydispersity index (PDI) on the developed formulations are displayed in Table 2. Results clearly show that the increase in the concentration of both GMO and CS was accompanied by an increase of the EE in the range of 81.77 ± 2.10% to 99.3 ± 1.25%.Moreover, NAR-HNPs prepared with GMO in addition to lipoid S45 exhibited the highest EEvalues, this may be possible due to higher concentration of the lipid 62 .Moreover, NAR-HNPs prepared with Lipoid S45 exhibited the highest EE-values.Concerning the PS of the prepared NAR-HNPS, increasing the concentration of CS led to a remarkable (p<0.05)increase in nanoparticle size.All the developed formulations had particle sizes ranging from 330.6 ±8.45 nm to 643.1 ±8.02 nm.Simultaneously, results show that both PS and ZP are increased in HNPs prepared with Lipoid S45 than comparable HNPs prepared without Lipoid S45.As depicted from the results, the surface charge of the formulations prepared with or without lecithin remained positive, ranging from 32.0 ±1.02 mV to 58.9±2.95mV.NAR-HNPs show low values for PDI ranging from 0.21±0.2 to 0.82±0.02.

In vitro release study
Figure 2 displays the release profile of various NAR-loaded NPs and free NAR.The results demonstrate that NAR has a slower release rate than the investigated NAR-HNPs.Comparing the studied formulations, NAR-HNP6 showed the highest percentage of NAR released up to 24 h.Furthermore, due to its high EE% and ZP, as well as its small PS and lowest PDI value, NAR-HNP6 was chosen as the optimum formulation for further investigation.The NAR-HNP6 DSC thermogram showed that neither NAR nor CS had any usual peaks.

Differential scanning calorimetry
Figure 3 illustrates the DSC thermograms of NAR, lipoid S45, P407, CS, and NAR-HNP6.At 256 °C, NAR displays a pronounced endothermic peak, attributed to drug melting and indicating crystallinity 63 .The DSC thermograms of P407 and CS revealed endothermic peaks at 61.2 °C and 210 °C, corresponding to their melting temperatures.Lipoid S45's amorphous nature prevented it from exhibiting any identifiable peaks.Table 2. Particle size, PDI, zeta potential, and entrapment efficiencies-values for the prepared NAR-HNPs.

pH measurement
According to literature, a pH range of 2-9 can be accepted for oral solution 64 .Therefore, the pH of an oral drug must fall within this range.The pH value of NAR-HNP6 is 4.9 ± 0.02 which is suitable for oral administration.

NAR-HNPs stability
Figure 5a-d depicts the impact of storage on EE%, PS, ZP, and PDI over 30 days at various temperatures, beginning on the first day of preparation.NAR-HNP stored at 4 °C did not show any indications of gelation, creaming, phase separation, or particle aggregation.However, an observed particle aggregation and creamy appearance were recorded at both temperature 25 °C and 40 °C.Additionally, NAR-HNP PS held at 4 °C did not change considerably (p = 0.9915) and displayed PDI values below 0.3 indicating a monodisperse pattern and a good homogenous population for the examined vesicles (p = 0.0973).On the other side, NAR-HNP stored at 25 °C and 40 °C exhibited an increase in PS and PDI accompanied with decrease in the entrapment efficiency (Fig. 5a-c).Additionally, as storage time was extended at 4 °C, the formulation absolute ZP value did not show significant   www.nature.com/scientificreports/change (p > 0.9999) (Fig. 5d), compared to that stored at 40 °C that demonstrated a significant decrease in the ZP values after 30 days of storage.

Verification method in HPLC
According to the presented data in Fig. 9, 10 the linearity of monoamines and NAR was in the optimum specification.Tables 3, 4, 5 depicts that the precision and accuracy of monoamines and NAR were in the optimum specification.Free NAR could be detected in plasma for up to 8 h.After oral administration of NAR-HNPs, C max were approximately 0.6697 ± 0.0673 µg/ml observed at 4 h in normal rats.It is quite evident that NAR-HNPs had faster absorption compared to free NAR.The increase in AUC by about 50% and the prominent increase in AUMC by about 185% revealed increased bioavailability.Peak serum concentrations of NAR-HNPs increased by about 33% compared to free NAR.t 1/2 of NAR-HNPs was prolonged about twofold than that of free NAR (10.26 ± 1.21 h for NAR-HNPs and 4.496 ± 0.2065 h for free NAR) and MRT was doubled (15.56 vs 8.326 h) indicating that free NAR was eliminated more slowly for NAR-HNPs than free Nar.NAR could be detected up to 24 h after oral administration of

Body weight alterations in rats administered NAR and NAR-HNPs in STZ-induced diabetes
Figure 12 demonstrates that the body weight of diabetic rats decreased by 18.01%, whereas NAR and NAR-HNPs reduced body weight gain by 10.63% and 4.92%, respectively (F = 133.8,p < 0.0001).GW9662 administration to NAR-treated diabetic rats markedly reduced body weight by 20.90% (F = 133.8,p < 0.0001).There was no

Behavioral changes in rats administered NAR and NAR-HNPs in STZ-induced diabetes
No remarkable difference was noticed between the basal and the correspondent final activity counts (F = 67.3,p < 0.0001), nor between the final activity counts of different groups of rats in the activity cage test (F = 3.452, p = 0.0022) (Fig. 13).
As shown in Fig. 14a, the immobility time of diabetic rats was significantly longer than that of normal rats by 6.08 times (F = 82.77,p < 0.0001).However, NAR and NAR-HNPs decreased the immobility time by 0.57 and 0.17 times, respectively (F = 82.77,p < 0.0001).In comparison to diabetic rats treated with NAR, the addition of

Discussion
The current study generates new evidence on the anti-depressant effect of NAR and NAR-HNPs on STZ-induced diabetic rats.To the best of the authors' knowledge this is the first study to demonstrate the role of PPARγ/NLRP3 pathway, cortical and hippocampal neurotransmitters in the anti-depressant effect of NAR and NAR-HNPs in diabetic rats.To uncover the involvement of PPARγ in the anti-depressant effect of NAR and NAR-HNPs, the effect of GW9962 was investigated.GW9962 was used to emphasize the involvement of PPARγ in the antidepressant effect of NAR and NAR-HNPs.The current findings prove that NAR-HNPs is more effective than NAR in mitigating depression in diabetic rats.
As depicted from this study, HNPs containing lipoid S45 displayed high EE% (>95%) compared to HNPs lacking lipoid S45.It was previously reported that increased drug encapsulation is more strongly impacted by lecithin incorporation 65 .This occurs when the phosphate groups in the negatively charged polar lecithin combine with the NH 3+ groups of CS, creating nanoparticles.Moreover, the increase in viscosity at higher concentrations, which causes the creation of larger droplets and ultimately larger nanoparticles, could be responsible for the observed increase in size with increasing CS concentration from 0.2 to 0.4% w/v 66 .Moreover, the interaction between the negatively charged lipoid S45 and the positively charged CS increases the diameter of HNPs 67,68 .The highly positive values for ZP observed for the HNPs prepared with lipoid S45 are also attributed to the electrostatic interaction of chitosan and lipids (Table 2).This finding is in harmony with prior study which demonstrates that during preparing the formulation of lecithin/chitosan hybrid nanoparticles, the cationic charge of chitosan dominate the anionic charge of lipoid 69 .At the same time, this recorded high ZP values play an important role on the stability of nanoparticles as particles in suspension will tend to reject each other and not agglomerate if all of the particles have the lowest negative ZP (above or equal to −30 mV) and the highest positive zeta potential (above or equal to +30 mV) 62 .As known, a PDI value less than 0.3 is regarded as acceptable, indicating a homogeneous population of nanoformulations 70,71 , which has already checked in NAR-HNP5 and NAR-HNP6.The lowest PDI value for NAR-HNP6 (0.21±0.02) is a good indication for the homogeneity of the system and proves a good distribution and little fluctuation in the PS 72 .
Regarding the in vitro release study, it is clear that increasing the concentration of chitosan from 0.2 to 0.4 % w/v decreases the release of NAR from the NAR-HNPs which can be returned to the interaction between the NAR and the CS resulting in retardation of the drug release.The more sustained release observed in NAR-HNP7 and NAR-HNP8 can be returned to the high CS concentration (0.4%w/v) beside the presence of lipoid S45 which exhibit a greater stabilization resulting in more release retardation 73 .On the other hand, NAR-HNP6 showed 75.2±2.24%NAR released after 24 h which makes it a good candidate to be chosen as the best formulation realizing the highest percent of NAR released compared to the other tested NAR-HNPs.
The NAR-HNP6 DSC thermogram revealed that there were no typical peaks for either NAR or CS, proving that the drug had been transformed into an amorphous form and that it had been incorporated into the NPs.As well, the TEM study shows how the particles are separated from one another which is a good indication of their Table 10.Relative expression of PPAR-γ genes in cortex and hippocampus of different experimental rats.The mRNA expression PPAR-γ is normalized with housekeeping gene (GAPDH), values are stated as means ± SEM, n = 3 per group (all samples run in duplicate and no template control were included in all runs).a vs normal group, b vs control STZ group, c vs STZ + NAR (50 mg/kg), d vs STZ + NAR-HNPs (50 mg/kg) at p < 0.05.NAR, naringenin; STZ, streptozotocin; NAR-HNPs, naringenin-loaded hybridized nanoparticles.stability 74 .The inner bright portion of the NPs represents the polymeric core encapsulating the drug, which is surrounded by a dark shell representing the lipid 69 .NAR-HNP6 exhibited an acceptable oral pH of 4.9±0.02and displayed a physicochemical stability at 4 °C.The pharmacokinetics evaluation showed that the novel formulation NAR-HNPs possesses superior absorption and elimination characteristics than free NAR.This was evident in the increase in C max and substantial prolongation of half-life and λz compared to free NAR.
Additionally, the increase of MRT points to increased drug retention in the systemic circulation; hence, providing an extended drug acting time.The observed increase in bioavailability, in terms of enhanced AUC and AUMC, in addition to enhanced MRT could be attributed to nanonisation that increases solubilization, and dissolution rate, thereby improving absorption in GIT and permitting efficient uptake in the intestine and prolonging drug retention at the absorption site 75 .
CS coating improves drug retention capability and cellular uptake through enhanced mucoadhesion to the mucin layer that delineates the superficial layer of enterocytes and M layers located in the intestine 76 .CS also improves oral absorption through inhibiting permeation-glycoprotein (P-gp) activity.P-gp is a membrane transporter that actively pumps drugs out of the enterocytes leading to decreased intestinal permeability and decreased oral absorption and bioavailability.By inhibiting P-gp activity, residence time of the drugs at active sites is prolonged, and hence absorption efficiency is increased 77,78 .GMO hinders enzymatic degradation during absorption and therefore reduces first-pass metabolism which is crucial for oral absorption 79,80 .In addition, entrapment into GMO enhances solubility of insoluble drug and thus facilitates their transport across cell membrane and consequently their oral absorption and oral bioavailability 81 .GMO also improve oral absorption via lowering P-gp activity 82 .The increase in the volume of distribution points to a higher distribution capacity of the formulation.Polymeric nanoparticles, have been found to be a promising solution to address a number of conventional issues, including low solubility, increased stability, a sharper size distribution, sustained and controlled release profiles, higher encapsulation efficiency for agents that are weakly water soluble, and the potential to improve bioavailability 83 .
In this study, 48 h following a single intraperitoneal administration of STZ in a dose of 50 mg/kg resulted in a marked elevation in blood glucose, body weight loss, depletion in serum insulin in addition to alteration in depressive-related parameters as FST, cortical and hippocampal monoaminergic contents.
The deficiency of monoamine neurotransmitters has emerged as a prominent area of investigation concerning the pathogenesis and treatment of depression.The main neurotransmitters in the brain are NE, 5-HT, and DA.NE is responsible for anxiety, alertness and attention whereas 5-HT affects the human mood and DA is related to motivation and reward 84 .In this study, STZ-induced depletion in cortical and hippocampal NE, 5-HT, and DA contents in addition to elevation in 5-HTAA and DOPAC contents.Those findings demonstrate the successfulness of the current depression-like model.
Moreover, STZ-induced depressive-like behavior was associated with oxidative stress evidenced by elevation in MDA and reduction in GSH, interference with PPAR-γ/NLRP3 pathway witnessed by reduction in cortical and hippocampal PPAR-γ expressions and elevation in both NLRP3 and IL-1β contents.Those findings are in line with prior studies which stated the involvement of oxidative stress and inflammation in the pathophysiology STZ associated depression 85,86 .
Anti-hyperglycemic effect of NAR and NAR-HNPs was witnessed by a reduction in blood glucose, elevation of serum insulin, and attenuation of body weight loss in comparison with diabetic rats and thereby improvement of the general health.As glycemic control plays a pivotal role in combating depression in diabetic patients, the anti-depressant effect of NAR and NAR-HNPs may be partly mediated via alleviation of hyperglycemia in diabetic rats.In line with this finding, NAR attenuated hyperglycemia in experimentally induced diabetic retinopathy and nephropathy in rats 87,88 .
Treatment with NAR and NAR-HNPs did not show any change in locomotor activity in the activity cage test, shortened immobility time and lengthened latency to immobility in FST in comparison with diabetic rats.Those findings reveal that the anti-depressant effect of NAR and NAR-HNPs was independent on changes in motor activity.In accordance, the anti-depressant effect of NAR has been previously reported in chronic unpredictable mild stress rat model 37 .This behavioral amelioration in NAR and NAR-HNPs-treated rats is in line with the noticeable increment in the content of neurotransmitters such as NE, 5-HT, and DA in the cortex and hippocampal region as well as a decrease in the levels of 5-HTAA and DOPAC metabolites in comparison to diabetic rats.In accordance, prior study showed that NAR reduced altered serotonergic, and dopaminergic neurotransmission in Alzheimer's disease rat model 89 .
NAR and NAR-HNPs anti-oxidant effect was evidenced by combating lipid peroxidation damage shown in the reduction of MDA contents in addition to promoting oxidative defense as revealed in the elevation of GSH contents in the cortex and hippocampus of diabetic rats.Therefore, their anti-depressant effect may be mediated via their anti-oxidant effect.In accordance, NAR exerted an anti-oxidant effect in the olfactory bulbectomyinduced depression model in mice 36 .It is well-established that hyperglycemia provokes oxidative stress and contributes to depression 90 .Therefore, NAR and NAR-HNPs anti-oxidant effect may have resulted from the mitigation of hyperglycemia.
Herein, NAR and NAR-HNPs significantly reduced NLRP3 and thereby IL-1β cortical and hippocampal contents in diabetic rats, revealing that the anti-depressant effect of NAR and NAR-HNPs could be partly linked to its anti-inflammatory effect.It is well-documented that oxidative stress accompanies inflammasome-mediated inflammation in depressed patients.NLRP3 activation results in the production of IL-1β, a pro-inflammatory cytokine that possess a crucial role in the progression of depression 91 .Also, previous studies reported that antidepressant treatment did not affect TNF-ɑ serum level but reduced IL-1β level, implicating the vitality of IL-1β in the treatment regimen 92 .Therefore, NAR and NAR-HNPs anti-depressant effect could have resulted from altering the oxido-inflammatory response.Consistent with the current finding, NAR showed anti-depressant effect by alleviating inflammatory response in mice subjected to hypoxic stress 35 .
Mounting evidence reported correlation between PPARγ and NLRP proteins 93 .Herein, NAR and NAR-HNPs significantly elevated PPARγ cortical and hippocampal gene expression compared to diabetic rats.Furthermore, GW9662 reversed NAR inhibitory effects on oxidative stress biomarkers and NLRP3 inflammasome activation.The current findings implicate the role of PPAR-γ/NLRP3 pathway in mediating the anti-depressant effect of NAR and NAR-HNPs in diabetic rats.In accordance, NAR increased PPARγ expression in experimental rat models of dementia and stroke 94,95 .

Conclusion
This study showed that NAR and NAR-HNPs possessed anti-depressant effect in diabetic rats, which could be interposed by mitigation of hyperglycemia, amelioration of oxidative stress and inflammatory response via PPARγ/NLRP3 pathway in addition to activation of monoaminergic system.GW9662 reversed those effects implicating the vital role of PPARγ in mediating NAR anti-depressant effect.NAR-HNPs showed the best antidepressant effect in diabetic rats.Lipid polymer HNPs made using the self-assembly method demonstrated improved NAR release profile, stability at 4 °C, good drug encapsulation efficiency and bioavailability with the required PS and PDI than free NAR.As the behavioral amelioration of NAR-HNP-treated rats in FST is in line with the distinguished elevations in cortical and hippocampal NE, 5-HT, and DA contents and reduction in 5-HTAA, HVA and DOPAC metabolites compared to NAR-treated rats.Thus, the loading of NAR in HNPs has a potent potential to be investigated as an anti-depressant therapy in DM.Further studies are necessary to elucidate the pathological alterations that may derive the anti-depressant effect of NAR-HNPs in diabetic rats.Clinical studies focusing on long term safety concerns, and delivery challenges are necessary to assess NAR-HNPs' effectiveness as a therapeutic agent for the treatment of depression in diabetic patients.Regarding future scope, it is important to explore the potential for personalized depression treatments by modifying nanoparticle formulations based on individual patient profiles, genetics, and drug responses.Also, it is essential to investigate the use of NAR-HNPs in combination with other anti-depressant drug to enhance treatment outcomes.

Figure 1 .
Figure 1.Schematic summary of the experimental study.

Figure 4
Figure 4 depicts the micrograph of NAR-HNP6.The image shows well dispersed black dots, round, nanosized, and homogeneous nanoparticles with a compact structure, and a smooth surface.

Figure 2 .
Figure 2. In vitro NAR release profiles of different hybridized nanoparticle formulations in comparison with the free drug in phosphate buffer saline pH 6.8 at 37 °C.

Figure 11
Figure 11 depicts NAR's mean plasma concentration-time profile after oral administration of the pure drug NAR and NAR-HNPs at doses of 50 mg/kg.Table6represents the pharmacokinetic analysis.At first glance, it is evident that free NAR is rapidly absorbed and eliminated with t 1/2 value 4.496 ± 0.2065 h and MRT was 8.326 ± 0.272 h.Free NAR could be detected in plasma for up to 8 h.After oral administration of NAR-HNPs, C max were approximately 0.6697 ± 0.0673 µg/ml observed at 4 h in normal rats.It is quite evident that NAR-HNPs had faster absorption compared to free NAR.The increase in AUC by about 50% and the prominent increase in AUMC by about 185% revealed increased bioavailability.Peak serum concentrations of NAR-HNPs increased by about 33% compared to free NAR.t 1/2 of NAR-HNPs was prolonged about twofold than that of free NAR (10.26 ± 1.21 h for NAR-HNPs and 4.496 ± 0.2065 h for free NAR) and MRT was doubled (15.56 vs 8.326 h) indicating that free NAR was eliminated more slowly for NAR-HNPs than free Nar.NAR could be detected up to 24 h after oral administration of

Figure 9 .
Figure 9. Linearity of NAR by HPLC verification method.

Figure 11 .
Figure 11.Plasma concentration-time curve of NAR after oral injection of NAR and NAR-HNPs.
Table 6 represents the pharmacokinetic analysis.At first glance, it is evident that free NAR is rapidly absorbed and eliminated with t 1/2 value 4.496 ± 0.2065 h and MRT was 8.326 ± 0.272 h.

Table 3 .
Precision results of monoamines in brain tissue.LOD: lower od detection; LOQ: Limit of quantification.

Table 4 .
Accuracy results of monoamines in brain tissue.

Table 7
reveals that diabetic rats manifested a dramatic elevation in blood glucose level to