Neuroprotective Potential of 6-O-(3-Hexadecyloxy-2-Hydroxypropyl)-Piperine-Nanoparticles in a Rat Model of Cerebral Ischemia-Reperfusion Injury

doi:10.21203/rs.3.rs-4813064/v1

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Research Article

Neuroprotective Potential of 6-O-(3-Hexadecyloxy-2-Hydroxypropyl)-Piperine-Nanoparticles in a Rat Model of Cerebral Ischemia-Reperfusion Injury

https://doi.org/10.21203/rs.3.rs-4813064/v1

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Purpose

Piperine is an alkaloid found in black pepper (piper nigrum) responsible for pungent smelling, potential therapeutic benefits. It has several significant biological properties, such as bioavailability enhancer, therapeutic potential, low water solubility, pharmakinetic properties, cardiovascular benefits, and neuroprotective effects. In a rodent model of transient focal cerebral ischemia-reperfusion injury (TFCIRI), piperine has been demonstrated to have a protective effect on the brain.

Methods

The present study was designed to prepare 6-O-(3-hexadecyloxy-2-hydroxypropyl dextran (HDD)--Piperine-Nanoparticles (HDD-PIP-NPs), its physicochemical characterization and neuroprotective potential against TFCIRI. The piperine was encapsulated in self-assembled 6-O-(3-hexadecyloxy-2-hydroxypropyl)-dextran (HDD) nanoparticles (HDD-PIP-NPs). HDD-PIP-NPs were characterized for their drug loading, entrapment efficiency, particle size, surface morphology, and in-vitro drug release profile.

Results

Plasma pharmacokinetics (C_max (4.71 ± 0.77), T_max (60 min), t_1/2 (449.19 ± 98.02), UAC_{1 − infinite} (641.62 ± 54.01), and UAC_{1 − 360} (641.62 ± 54.01)) brain distribution profile of PIP in cerebrum, cerebellum and cortical region, and the neuroprotective potential of HDD-PIP-NPs have been characterised in rat model of TFCIRI. HDD-PIP-NPs (14, 28, and 56 mg/kg) was administered orally after 1h onset of TFCIRI. HDD-PIP-NPs chemical synthesis and its biochemical and biophysical characterization have been done. The percentage decreased infarction (~ 52.52%) of orally administered HDD-PIP-NPs (56 mg/kg) is ameliorated in rat model of TFCIRI. Physiological parameters such as CBF (~ 201.23), pCO₂ (~ 38.55 mmHg), pO₂ (~ 128.42 mmHg) and rectal temperature (~ 36.9ºC) was recorded the 56mg/kg oral treatment. Neurological deficit score recorded after the 24h, 48h and 72h of reperfusion injury. Brain penetration studies and bioavailability of PIP estimated in male Charles foster albino rats. The results indicated that HDD-PIP-NPs treatment significantly decreased the percentage of infarction, percentage brain water content and neurological deficit scores in dose dependent manner.

Conclusion

These findings suggest that piperine (PIP) possesses significant neuroprotective potential. Its nano-formulation, HDD-PIP-NPs, has shown enhanced plasma and brain pharmacokinetics, leading to improved neurological outcomes following transient focal cerebral ischemia-reperfusion injury (TFCIRI) in rats. HDD-PIP-NPs could potentially be useful for neuroprotective interventions against various neurological disorders.

Piperine-Nanoparticle

Dextran

Entrapment efficiency

transient focal cerebral ischemia-reperfusion injury

Pharmacokinetics

The ischemic stroke, which records for around 80% of all strokes, is the fifth driving reason for mortality and main cause for adult disability with significant clinical and socio-economic burden (Addo et al. 2012, Smith et al. 2008, Donkor 2018). The current therapies intravenous thrombolysis (IT) and endovascular thrombectomy (EVT) are evidence-based treatments for adults with ischemic stroke, re-establishing cerebral blood flow within narrow time window in order to prevent damaging the "penumbra" which surrounds the infarct core (Smith et al. 2008; Wu et al. 2018). It remains the key treatment around the world, however not perfect due to compelling to 10% or a smaller number of patients treated within the appropriate therapeutic time window. Over the last few decades, herbal medication has been existing as an alternative scheme for the development of novel drugs that can be used in CNS disorders including cerebral stroke (Suk 2005). Among all plant-derived bioactive phytochemicals, piperine a nitrogenous alkaloid compound of black pepper (Piper nigrum Linn.), longum pepper (Piper longum Linn.) fruits has drawn special attention (Tripathi et al. 2022, Quijia et al. 2021). PIP has a wide range of pharmacological applications such as antimicrobial, anti-cancer, anti-oxidant, anti-ischemic and anti-cognitive impairment (Capasso et al. 2002, Singh and Kumar 2007, Singh and Kumar 2017). Also, PIP administration delayed seizure onset and decreased neurological impairment of various neurological murine models (Vaibhav et al, 2012, da Cruz et al. 2013, Hua et al. 2019). Despite such beneficial advances in the therapeutic application, the clinical role of native PIP is hindered due to low water solubility, good lipid solubility, poor bioavailability, rapid metabolism, and poor pharmacokinetics. However, earlier pharmacokinetic profiling and brain uptake of PIP is performed to explore various routes of administrations (Cherniakov et al. 2017, Ren et al. 2018). Recent experimental evidence suggests that the encapsulation of hydrophobic drugs in biodegradable polymers has opened new avenues in nanomedicine (Anissian et al. 2018). Dextran is broadly exploited in biomedical applications and as a constituent of drug-delivering nanoparticles (Wasiak et al. 2016). The major benefits for dextran as a nano-carrier are super solubility in water, minimum cellular toxicity and renal failure, high loading capacity and intrinsic viscosity, and short storage period (Wasiak et al. 2016). Furthermore, the aim of the present work was to investigate the neuroprotective potential of PIP-HDD-NPs against the rat model of TFCIR. The following objectives were set to achieve the above-mentioned aim: (1) preparation and characterization of HDD-PIP-NP, (2) Plasma pharmacokinetics and brain distribution, (3) neuroprotective assessment of HDD-PIP-NPs in middle cerebral artery occlusion (MCAO) rat model.

2.1. Drugs and chemicals

Dextran (Molecular Weight 298 kDa), Piperine, and 2, 3, 5-Triphenyl tetrazolium chloride (TTC), were purchased from Sigma Aldrich (St. Louis, USA). Other reagents and chemicals obtained from local firms (India) were of maximum purity. Size of nanoparticles was measured on Zetasizer Nano-ZS (Malvern Instruments, UK). The water, acetonitrile and acetic acid HPLC grade were purchased from TCI (India). Heparin sodium salt was purchased from porcine. All other chemical used in this work were HPLC grades.

2.2. Synthesis of HDD-PIP-NPs

PIP was encapsulated in 6-O-(3-hexadecyloxy-2-hydroxypropyl)-dextran (HDD) following the protocol reported with certain modifications (Ray et al. 2013). HDD (0.250 g) was suspended in 60 mL double distilled water and to this, a PIP solution (0.025 g dissolved in 8 mL of ethanol) was added over a time course of 15 min. The solution was stirred at 300 rpm for 24 hours in the dark at a temperature of 25 ± 2 ºC. Subsequently, the resulting solution was dialyzed against double distilled water with stirring for 24 hours, with regular changes of water to remove impurities. 24 h was enough to release unbound PIP which has been found in a controlled experiment. The dialyzed solution was lyophilized for 24 h to obtain HDD-PIP-NPs (0.210 g) as a white solid in ~ 67% yield.

2.3. Characterizing the HDD-PIP-NPs

2.3.1. Percent yield

The yield (%) of HDD-PIP-NPs was estimated by following formula:

Yield (%) = (Weight of HDD-PIP-NPs X 100)/(Weight of (PIP + HDD)………Equation-(1)

2.3.2. Particle size measurement

The average size and polydispersity index (PDI) of the HDD-PIP-NPs were measured using dynamic light scattering (DLS) (Clayton et al., 2016). The freeze-dried NPs were dispersed in water at a concentration of 1 mg/mL, and the particle size was subsequently measured. The particle size measurements were carried out at 25 ± 2º C with the following parameters in Zetasizer Nano Instrument: 10 measurements per sample; nominal 5mW He-Ne laser operating at 633 nm wavelength; absolute viscosity for water 0.89 centipoises (cP), and refractive indices of water and dextran, 1.33 and 1.36, respectively. The size measurements were done in triplicates.

2.3.3. Drug loading and entrapment efficiency

The PIP loading content and its entrapment efficiency (EE) of PIP-NP were determined by centrifugation at 5000 rpm for 20 min to separate the unentrapped PIP. After the centrifugation step, added the appropriate amount of methanol for ultrasonic and then used HPLC (LC-20AD, CBM-20A, CTO-20AC, Rheodyne injector, and SPD-20A). The PIP loading and entrapment efficiency were determined by spectrophotometrically using Lambda 20 UV/VIS Spectrophotometer (Perkin Elmer, USA) (Alfei et al. 2020). The %DL and %EE was estimated using the formulas as given below. All the measurements were performed thrice.

%DL =(Weight of PIP in HDD-PIP-NPs X 100)/ (Weight of HDD-PIP-NPs).Equation-(2)

%EE = (Amount of PIP present in the HDD-PIP-NPs X 100)/(Amount of PIP taken).

….Equation-(3)

2.3.4. Surface morphology of nanoparticles

The surface morphology characterization of the HDD-PIP-NPs was performed using TEM.

Transmission Electron Microscopy

In brief, lyophilized powder of HDD-PIP-NPs was dispersed in water (5mg/mL) and a drop was placed on a surface of TEM grid and a drop of 1% uranyl acetate was added to the surface of the Formvar-coated grid. The excess fluid was removed after 1 min and the grid surface was air dried at 25 ± 2 ºC before being loaded into TEM instrument. HDD-PIP-NPs were visualized at 80 kV by Gaten digital micrograph (PA, USA).

2.4. PIP Release study from HDD-PIP-NPs

For drug release profiling from HDD-PIP-NPs, approximately 5 mg of the nanoparticles was dispersed in 1 mL of PBS (pH 7.4). This dispersion was then transferred into a dialysis tube with a 12 kDa molecular weight cut-off, which was suspended in 20 mL of PBS in a glass vial. The solution was stirred at 240 x g at 37°C. 200 mL samples were collected from the glass vial at pre-determined intervals and analyzed spectrophotometrically at 345 nm and at the same time, an equal quantity of fresh buffer was added to the glass vial and the release process was continued. The amount of piperine released was determined by referencing a standard curve of pure piperine in PBS that had been previously generated.

2.5. Preparation of Piperine Test Formulations

For oral administration, 350 mg of HDD-PIP-NPs were dispersed in 62.5ml normal saline (0.9% and pH 5.5) strength to obtain the concentration of 5.6 mg/ml, maintaining the dose 56 mg/kg and a dose volume of 10ml/kg prepared by dispersing 350mg HDD-PIP-NPs. The prepared solutions were protected from light and incubated at 4°C. The prepared HDD-PIP-NPs dispersion was vortex for 30 seconds and immediately used for dosing.

The PIP (56mg/kg) with dose volume (5.6mg/ml) dissolved in 0.1M phosphate buffer (pH 7.4) given orally.

2.6. Chromatographic system

PIP concentration in the relevant experiments were analysed at 340 nm using a High-performance liquid chromatography (HPLC) system from Shimadzu Scientific Instruments, USA. The system, consisting of a chromatographic pump (LC-20AD), System Controller (CBM-20A), Column oven (CTO-20AC), Rheodyne injector, and UV–Vis detector (SPD-20A), was operated at the room temperature (24 ± 1℃). Data collection, calibration, and integration LC Solutions chromatography data analysis system was used to collect, calibrate and integrate data. Kinetex Reversed-Phase C₁₈ column (250 x 4.6 mm, particle size 5µm) was utilized for the separation. Acetonitrile, water, and acetic acid were mixed at the ratio of 60:34.5:0.5(v/v/v), and the solution was further filtered with a Millipore filter system (0.22µm)to obtain the mobile phase and the flow rate was maintained at 1.5 ml/min (Ren et al., 2018). The pH of the mobile phase was adjusted to 3.0.The mobile phase was degassed for 30 minutes after filtration and before being utilized for the experiments.

2.7. Preparation of Standard Solutions and Quality Control (QC) Samples

Methanol extracted blank plasma was used for the preparation of HDD-PIP-NPs stock solution (1000 µg/ml). The stock solution was further diluted using blank plasma to obtain working solutions of concentration (1.00, 5.00, 10.00, 50.00, 100.00, and 500.00 µg/ml). The range of concentrations (0.5, 2.5, 5, 25, 50, 250 µg/ml) were prepared for obtaining the standard curve. The solutions were prepared by diluting the working solution with blank plasma, cortex, cerebellum, hippocampus, and CSF (1:1). The calibration standard was prepared by spiking 10µl of working solutions. For determining the accuracy and precision of the HPLC method quality control (QC) samples of high (250.00 µg/ml), medium (5.00 µg/ml), and low (0.5 µg/ml) concentrations were prepared. All samples were light protected and stored at 4℃.

2.8. Pharmacokinetic Study

Four male Charles Foster rats were taken for oral gavage (n = 4). All rats received a 56mg/kg dose of HDD-PIP-NPs via oral gavage. Heparinized capillary tubes were used to collect blood (0.2 ml) from the retro-orbital plexus at following time intervals: 0 (pre-dosing), 15, 30, 60,120, 240, 360, 480, and 1440 min after oral administration in accordance to the study by Kumar et al (Kumar et al., 2019). The sampled blood was stored in labelled tubes containing sodium heparin. The collected blood samples were centrifuged at 4000 rpm for 10 min at 4°Cand the separated plasma was transferred to labelled Eppendorf tubes. The obtained plasma samples were stored at − 80°C till they were further analysed by HPLC (Kumar et al. 2019). The relative bioavailability of PIP and HDD-PIP-NP was calculated according to the equation:

Relative BA (%) = 100 X \(\:\frac{{AUC}_{formulation}/{Dose}_{formulation}}{{AUC}_{PIP\:or\:HDD-PIP-NP}/{Dose}_{PIP\:or\:HDD-PIP-NP}}\)…………Equation-(4)

The pharmacokinetic results are represented as mean ± SE. The time to reach the maximum plasma concentration (tmax) and peak plasma concentrations (Cmax) values were measured from the concentration-time data of plasma. Other pharmacokinetic parameters such as elimination half-life (t1/2), the area under the curve from time zero to twenty-four hours (AUC0-24hr), the volume of distribution (Vz), mean residence time (MRT last), and Total body clearance (Cl) were calculated by non-compartmental methods using software PK Solver.

2.9. Brain Distribution Study

Twenty (20) male Charles Foster rats were used for oral administration. All rats were further divided into five groups (n = 4 for each group), for the time-points of 30 minutes, 60 minutes, 120 minutes, 240 minutes and 360 minutes. Anaesthesia and dosing were performed similarly to the pharmacokinetic study. The samples were isolated at the time interval 30, 60 120, 240, and 360 minutes after HDD-PIP-NP oral dosing. Following cervical dislocation, the brains were isolated and cleaned with normal saline. Three regions of the brain samples (cortical, hippocampus, and cerebellum) were carefully isolated and immediately processed for the HPLC analysis.

2.10. Plasma and Brain Sample Preparation

Collected plasma samples were mixed with HPLC grade methanol (1:2) by vortexing thoroughly for 30 seconds and a clear supernatant was obtained by centrifuging the mixture at 3000 rpm for 10 min at 4°C. To estimate the PIP concentration for each sample, 10 µl of each obtained clear supernatant was used for HPLC analysis.

The different brain regions were homogenized in methanol using a tissue homogenizer (BR Biochem Pvt. Ltd, India) in an ice cold bath. For the homogenization of cerebellum (0.5gm), cortex (0.5gm), and hippocampus (20 mg) 1, 1, and 0.5ml of methanol were utilized respectively. Subsequently, the brain homogenate–methanol mixture was centrifuged at 5000 rpm for 20min at 4°C and the clear supernatant was collected.10 µl of each sample was used for further analysis with HPLC.

2.11. Experimental Animals Grouping

All experimental procedures and surgeries were conducted in accordance with the animal use protocol approved by the Central Animal Ethical Committee at the Institute of Medical Sciences, Banaras Hindu University, Varanasi (Registration No. 542/GO/ReBi/S/02/CPCSEA). Inbred male Charles foster (CF) albino rats (250 ± 30 g body weight) were acclimatized for 14 days by maintaining 12h light/dark cycle, stable humidity, ambient temperature (25 ± 2°C) and were provided with pellet diet and water.

The animals were fasted for 8–10 hours before the experiment, with ad libitum access to water.

Group I: TFCIR surgical procedure only (normal saline as vehicle only; 2 hrs. post-reperfusion) (n = 6) for 3 days.

Group II: TFCIR surgical procedure with HDD-PIP-NPs (14 mg/kg, equivalent to 8.75 mg/kg bulk PIP, 2 hrs. post-reperfusion) (n = 6) for 3 days.

Group III: TFCIR surgical procedure with HDD-PIP-NPs (28 mg/kg, equivalent to 17.5mg/kg bulk PIP, 2 hrs. post-reperfusion) (n = 6) for 3 days.

Group IV: TFCIR surgical procedure with HDD-PIP-NPs (56 mg/kg, equivalent to 35mg/kg bulk PIP, 2 hrs. post-reperfusion) (n = 6) for 3 days.

2.12. Surgical procedure for Transient Focal Cerebral Ischemia Reperfusion (TFCIR)

Cerebral MCAO surgery was performed as described previously with minor modifications (Tripathi et al. 2014, Tripathi et al. 2021). Briefly, rats at the age of 6–8 weeks were anesthetized with a cocktail of ketamine (140 mg/kg) and xylazine (23.32 mg/kg), intraperitoneal (i.p.) injection. The initial step of MCAO surgery involves cleansing the neck region of the animal with 70% ethanol. Following this, the skin is incised to expose and isolate the right common carotid artery. The right common carotid artery was exposed at the level of the external and internal carotid artery (ECA and ICA) bifurcation. A 25 mm length and 0.22mm diameter of 4.0 siliconized monofilament nylon suture (Doccol corporation) was inserted into ECA from the common carotid artery bifurcation and pushed into the ICA for 10 mm until a slight resistance was felt, to block the origin of the middle cerebral artery (MCA). Following this, the skin incision was sutured, and reperfusion was initiated 60 minutes after MCAO by slowly withdrawing the suture thread until the tip cleared the internal carotid artery (ICA). After the procedures, animals were placed back into their cages and closely monitored. Their body temperature was maintained at (37 ± 1.0) °C using a heating pad (Far infrared warming pad, Kent Scientific Corporation).

2.13. Brain Infarction, Brain water content and Neurological score analysis

Brain infarction was analysed at day 3 using TTC staining procedure (Schilichting et al. 2004). Briefly, 2mm coronal section of each rat brain was incubated in 2% TTC solution for 30 minutes. 10% formalin was used to fix the stained brain slices and pictures were taken by the scanner. Percent infarction was measured and calculated according to (Tureens et al. 2004). The percentage brain water content of all groups was determined at day 3 by the wet-weight and dry-weight method (Keep et al. 2012) using the formula:

% Brain water content = 100 X (Wet Weight - Dry Weight)/ (Wet Weight). Equation-(5)

The rats were decapitated under chloroform anaesthesia, followed by isolation of the brains from the skull which was weighed immediately and the corresponding weight was noted as weight wet. The dry weight was measured after drying the brain in a desiccating oven at 100°C for 2 days to obtain a constant weight (Kumar et al. 2019).

Neurological deficits scores were calculated at 24 h, 48 h and 72 h after reperfusion (n = 6) by the method of Longa (Tripathi et al. 2014). The five-point scale was defined as follows: Grade 0 indicated no neurological deficits; Grade 1 indicated failure to extend the contralateral forepaw; Grade 2 indicated circling to the ipsilateral side; Grade 3 indicated falling to the contralateral side due to brain damage; and Grade 4 indicated no spontaneous walking and depressed consciousness.

Data analysis

Statistical analyses were conducted using GraphPad Prism version 5 (San Diego, CA). The data, presented as mean ± SD, were assumed to follow a normal distribution. Statistical significance was determined using One-Way analysis of variance (ANOVA), followed by Tukey’s multiple-comparison test. Neurobehavioral scores, expressed as median (range), were analyzed using the Mann − Whitney U test. A p-value of less than 0.05 was considered statistically significant.

3.1. The synthesis and characterization of hexadecyl dextran-doxorubicin-piperine nanoparticles (HDD-PIP-NPs) were conducted, followed by an investigation of the in vitro piperine (PIP) release profile

Hexadecyl dextran-doxorubicin-piperine nanoparticles (HDD-PIP-NPs) were prepared using the nanoprecipitation method, employing the biodegradable and biocompatible polymer dextran (Ray et al. 2013). HDD-PIP-NPs were prepared by combining piperine and the polymer at a ratio of 1:10 in double-distilled water at a temperature of 25 ± 2 ºC. The polysaccharides substituted with 3-(hexadecyloxy)-1-chloropropan-2-ol achieved a PIP loading with an encapsulation efficiency of 67%, resulting in a percent drug loading (%DL) of 3.4%. It was hypothesized that PIP interacted with the C-16 chains through physical interactions within the self-assembled substituted polysaccharides. The average hydrodynamic diameter/particle size of HDD-PIP-NPs, as measured by DLS, was 97.8 ± 8.5 nm, indicating a low polydispersity index (0.18) and the formation of nearly monodisperse nanoparticles (Figure 2). The surface morphology of the HDD-PIP-NPs was analyzed using transmission electron microscopy (TEM). The average nanoparticle size determined by TEM ranged from approximately 19 to 30 nm (Figure 2A). Overall, the nanoparticles exhibited a spherical shape with smooth surfaces. The particle size measured by TEM was notably smaller than that determined by DLS. This phenomenon might be due to the fact that DLS measures the hydrodynamic diameter of the NPs, where the HDD-PIP-NPs were surrounded by water molecules. However, for transmission electron microscopy (TEM) studies, the HDD-PIP-NPs were in a dry powder state. The release kinetics was evaluated in a phosphate-buffered saline (PBS) solution at a pH of 7.4 and a temperature of 37±2ºC (Figure2). A burst release of approximately 39% of PIP from the HDD-PIP-NPs occurred within the first 6 hours, followed by a sustained release of PIP observed over the next 8 days.

3.2. Chromatography procedure validation

Chemically, the PIP molecule consists of conjugated aliphatic chains that serve as a connecting structure between the piperidine and the 5-(3,4-methylenedioxyphenyl) moiety (Tripathi et al. 2022). Piperine contains amide and carbonyl group, insoluble in water and has weak basic character that hydrolyse in water in piperic acid and piperidine attributes to the acidity of the compound leading to the necessity of an acidic mobile phase (pH 3.0) for proper retention and separation of the compound in reversed-phase chromatography (Tiwari et al.2020). An acidic mobile phase also ensures less interference of the polar compounds present in plasma. The retention time of HDD-PIP-NPs had a retention time of 4.2 mins at 1.5 ml/min flow rate. The linear calibration curve was plotted as area vs. Concentration for the range of 0.0500–50.00 μg/ml of HDD-PIP-NP in rat plasma and the standard curve had a mean correlation coefficient of r= 0.99881. The HPLC method’s accuracy and precision were assessed with QC samples at HDD-PIP-NP concentrations ranging from 0.05 to 50μg/ml. The limit of quantification (LOQ) of the present HPLC method was 0.05μg/ml. Following successful validation, the method was subsequently applied to assess PIP levels in both the plasma and brain of rats (Table 1).

Table-1: Here are the details of the HPLC parameters for calibration, accuracy, and precision assessment of HDD-PIP-NPs: Calibration: A standard calibration curve was constructed using known concentrations of HDD-PIP-NPs. Accuracy: The accuracy of the method was determined by comparing the measured concentrations of HDD-PIP-NPs with their known concentrations. Precision: The precision of the method was evaluated by analyzing multiple replicates of the same sample and calculating the relative standard deviation (RSD) of the results. The HPLC method was validated according to these parameters to ensure reliable quantification of HDD-PIP-NPs.

3.3. Plasma pharmacokinetics of PIP after single dose of HDD-PIP-NPs

Pharmacokinetic results revealed that the maximum concentration of PIP in plasma 4.71±0.77µg/ml at 60 minutes after administration of 56 mg/kg single dose of HDD-PIP-NPs. Figure 3 represents the change of piperine concentration in the plasma over the defined time rangedue to oral administration of HDD-PIP-NPs. The pharmacokinetic parameters of HDD-PIP-NPs were determined using the softwarePK Solver and are listed in table 2.

3.4. Pharmacokinetics of piperine in the brain after a single oral dose of HDD-PIP-NPs

The concentration of PIP in cortex, cerebellum and hippocampus were determined at 30, 60-, 120-, 240- and 360-minutestime intervals (Figure 4). The C_maxvalue of PIP in cortex, cerebellum and hippocampus were 7.01µg/ml, 4.76µg/ml, and 3.75µg/ml at 120 minutes respectively. The brain pharmacokinetic profile of HDD-PIP-NPs after single oral administration (56mg/kg) is presented in Table 3. The exposure of PIP (AUC _0-360) was higher in cortex compared to cerebellum and hippocampus.

Table 3: Brain pharmacokinetic profile of HDD-PIP-NPs (56mg/kg) after single oral administration

3.5. HDD-PIP-NPs protect against TFCIR induced brain injury in rat

The % infarct area of the treated group with HDD-PIP-NPs was significantly decreased in a manner dependent on the dosage (Türeyen et al. 2004). At dose 56 mg/kg, the % infarction area was decreased from 36.85 ± 3.99% to 17.50 ± 3.42% (p < 0.05) (Figure 4a). Brain edema refers to the abnormal accumulation of fluid in the brain, leading to an increase in brain water content. Increased fluid puts pressure on the brain tissues, which can lead to a range of symptoms and potentially serious complications. This increase brain water content can disrupt the delicate balance of ions and other substances in the brain affecting its normal functioning. Treatment with 56 mg/kg HDD-PIP-NPs significantly reduced the brain water content as compared to the vehicle-treated group (p<0.05). There were no notable differences observed between the vehicle-treated group and the group treated with 14 mg/kg HDD-PIP-NPs, however there are no differences between the groups treated with 14 mg/kg and 28 mg/kg HDD-PIP-NPs. Another set of experiments conducted to see the effect of HDD (66mg/Kg), PIP (66mg/kg), and HDD-PIP-NPs (56 mg/Kg) in animals. The administration of 56 mg/kg HDD-PIP-NP notably restored the brain water content close to its normal value (Figure 4b). After reperfusion, the neurological deﬁcits scores were evaluated at 24 hrs, 48 hrs, and 72 hrs. Figure 4C illustrated the neurological deﬁcit score of 56 mg/kg HDD-PIP-NPs treated group were signiﬁcantly decreased compared to the vehicle group. Therefore, 56 mg/kg HDD-PIP-NPs (35 mg/kg dose of PIP) was an effective therapeutic strategy against TFCIR injury (Figure 4c).

The earlier report suggested the cytoprotective use of nanoparticles to investigate the biological activities in various disease animal models (Kumar et al. 2019). The utilization of natural phytochemicals from plant sources could be a sustainable solution for the synthesis of nanoparticles (Srivastava et al. 2020). The main alkaloid found in black pepper, known as PIP, demonstrates powerful neuroprotective effects in a variety of neurological models (Tripathi et al. 2022). However, the low water solubility of PIP (40 mg/l), unclear pharmacokinetics properties in human and potential toxicity during the long-term use at higher doses has been limited its clinical application. In this study, piperine was loaded on 3-(hexadecyloxy)-1-chloropropan-2-ol dextran and its protective effect was evaluated against TFCIR in rats. Dextran nano formulation is widely used to deliver the drug across the blood brain barrier (Wasiak et al. 2016). Nano-formulation of Octylglyceryl dextran-graft-poly (lactic acid) was demonstrating potential for peptide delivery into the human brain endothelial cells (Boussahel et al. 2017). Cell culture based studies demonstrated that alkylglyceryl-modified dextran-graft-poly nano formulation is a potential candidate for drug delivery to the brain (Toman et al. 2015). Solid lipid nanoparticles of dextran sulfate complex were also shown improved vincristine delivery to the brain (Aboutaleb et al., 2014).

In the present work, receptor targeted 6-O-(3-hexadecyloxy-2-hydroxypropyl dextran (HDD) loaded PIP were characterised by their entrapment efficiency, percent yield, drug load, surface morphology, particle size and in-vitro drug release (Ray et al. 2013). Synthesis of amphiphilic hexadecylated polysaccharide is a two-step process. The first step involves reaction of hexadecyl alcohol with epichlorohydrin by epoxide ring opening reaction, thus producing 3-(hexadecyloxy)-1-chloropropan-2-ol. 3-(hexadecyloxy)-1-chloropropan-2-ol is then converted into its epoxide in-situ in the presence of an alkali and reacts with the hydroxyl groups of polysaccharides (dextran) leading to formation of O-(3-(hexadecyloxy-2-hydroxypropyl) substituted polysaccharides. Furthermore, incubation of dextran substituted O-(3-(hexadecyloxy-2-hydroxypropyl) with PIP to synthesize the HDD-PIP-NPs. Non-covalent interaction such as hydrogen bonding and Vander Val force of interactions are involved between the chemical reaction of HDD and PIP molecules. The brain uptake and pharmacokinetics studies of HDD-PIP-NPs was performed according the protocol of Ren et al., with 35 mg/kg single oral dose and HDD-PIP-NPs exhibited similar pharmacokinetic profile in plasma and brain (T_max = 4h) (Ren et al. 2018). We performed brain penetration studies to evaluate the brain and plasma pharmacokinetics profile of HDD-PIP-NPs. Furthermore, results revealed that the HDD-PIP-NPs can cross the BBB after oral administration and distributed into various regions of brain cortex, cerebellum and hippocampi. The maximum concentration of PIP reached in cortex, cerebellum and hippocampi was 7.01 ± 0.97µg/ml, 4.76 ± 0.24 µg/ml, and 3.75 ± 0.50 µg/ml respectively after single oral administration of HDD-PIP-NPs (56mg/kg) (Fig. 4). In the present study, the pharmacokinetic profile of piperine (PIP) in the brain and plasma, when administered as HDD-PIP-NPs nanoparticles (NPs), demonstrated distinct and enhanced results. It is evident that when HDD-PIP-NPs were encapsulated in dextran polysaccharide its T_max value was decreased to 1 h for plasma pharmacokinetic and 2 h for the brain pharmacokinetics. To investigate the comparative effects of various components used in Nanoparticle synthesis, such as HDD, dextran, and PIP, we also examined their neuroprotective properties against TFCIRI (Fig. 5). Rapid remedial after ischemic insult is the critical strategy for the development of neuroprotective environment. We observed that the 56 mg/kg dose of HDD-PIP-NPs had a significant neuroprotective effect against TFCIRI induced animal model (Fig. 4). TTC staining is widely accepted procedure to determine the brain infarction (Kramer et al. 2010). In this study, treatment with 56mg/kg HDD-PIP-NPs significantly reduced the % brain infarction (≈ 17%) compared to saline group (≈ 36). Similarly, % brain water content was restored in 56mg/kg treatment group compared to saline group. The PIP also ameliorated the neurological deficit score outcomes in 24 h, 48 h and 72 h survival time. These finding suggest that, HDD-PIP-NPs has neuroprotective potential, demonstrated by infarction estimation, neurological outcomes evaluation and to improve the plasma and brain pharmacokinetics profiles in rat model of TFCIRI.

The study likely involves chemical synthesis of HDD-PIP-NPs (6-O-(3-hexadecyloxy-2-hydroxypropyl dextran (HDD)--Piperine-Nanoparticle) and tests their ability to protect the brain from ischemic injury caused by TFCIR, a type of rodent stroke model. Charles Foster albino rats are a commonly used animal model in medical research. Dextran based PIP-NPs is widely exploited in medical products and as a component of drug-delivering phytochemicals. Here, we tested whether HDD-PIP-NPs efficiently penetrate and homogeneously distributed in the brain at a similar pharmacokinetic profile in each region. Also, brain penetration studies suggest that the oral route of HDD-PIP-NPs delivery could be efficient in neuroprotection and drug delivering to the target site. HDD-PIP-NPs enhanced the therapeutic effects of PIP on TFCIR in rats, including decreasing infarction, improving behaviour scores. Our current research finding suggests that HDD-PIP-NPs can improve the brain uptake of PIP. The present study will also help in further investigations regarding the correlation between the pharmacokinetic and therapeutic intervention of HDD-PIP-NPs in other neurodegenerative disorders. The mechanism of action of HDD-PIP-NPs involves the stabilization of cell membranes, which helps to prevent neuronal damage and cell death. One study found that HDD-PIP ameliorated the extent of brain damage in rats subjected to tMCAO, a model of ischemic stroke. The biomedical researchers observed that HDD-PIP-NPs treatment resulted in reduced brain swelling, decreased infarction, and improved neurological function compared to untreated animals. However, it should be noted that while HDD-PIP-NPs has shown promise in preclinical studies, further research is needed to determine its safety and efficacy in humans.

HDD-PIP-NPs

3-(hexadecyloxy)- 1-chloroprocaine-2-ol dextran-piperine-nanoparticle

TFCIR

transient focal cerebral ischemia-reperfusion

MCAO

middle cerebral artery occlusion

DMF

dimethyl fumarate

PBS

Phosphate buffer saline

TTC

2,3,5-triphenyl tetrazolium chloride

HPLC

High-performance liquid chromatography

T_max

maximum plasma concentration

C_max

peak plasma concentrations

t_1/2

elimination half-life

AUC0-24h

area under the curve from time zero to twenty-four hours

the volume of distribution,MRT_last,mean residence time

Total body clearance.

Human Ethics and Consent to Participate

Funding

This work was supported by Department of Science and Technology-Science and Engineering Research Board (PDF/2016/002996/LS).Author AK Tripathi has received research support from DST-SERB.

Author Contribution

#Dr. Amit Kumar Tripathi: Initiated project, Performed major experiments, brain and blood sample collection, TFCIRI Surgery, and wrote the full Manuscript. #Dr. Lipika Ray: Performed piperine Nanoparticle synthesis and write her chemical synthesis of Nanoparticle. #Dr. Gaurav Kumar: Performed the pharmacokinetics and brain penetration studies. #Dr. Sumedha Mukherjee: Performed dosing and collection of blood samples. #Dr. Sunil Kumar Mishra: Continuous help to performed experiments andhelp in writing the discussion properly. #Dr. Ranjana Patnaik: Provide the extensive suggestion for designing the title and subtitle, and motivation for the project to conduct. Electrophysiology Lab is provided with the Funding from Indian Institute of Technology.

Acknowledgement

Data Availability

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Table 1. Plasma pharmacokinetic parameters of HDD-PIP-NPs (56mg/kg administered) after single oral administration.

PK parameter	Plasma
Dose	35mg/Kg (after release)
C_max (µg/ml)	4.71±0.77
T_max (min)	60±0.00
AUC _0-1440 (μg min/ml)	593.47±72.22
AUC _0-∞ (μg min/ml)	641.62±54.01
t_1/2(min)	449.19±98.02

Data are presented as mean±S.D. (n= 4).

Table 2: HPLC method parameters for calibration, accuracy and precision of HDD-PIP-NPs.

Sample	R	P Value	RSD (%) of Lowest QC sample Concentration (0.05 µg/ml) (N=6)		RE (%) of Lowest QC sample Concentration	Average Percentage recovery (%) of three QC samples	LOQ (µg/ml)	LOD (µg/ml)
Sample	R	P Value	Intra-day	Inter-day	RE (%) of Lowest QC sample Concentration	Average Percentage recovery (%) of three QC samples	LOQ (µg/ml)	LOD (µg/ml)
Plasma	0.9999	<0.0001	4.1	5.3	7.1	102.35	0.052	0.013
Cortex	0.9999	<0.0001	5.2	7.0	6.2	97.22	0.056	0.018
Cerebellum	0.9997	<0.0001	6.4	8.5	6.4	98.73	0.060	0.021
Hippocampi	0.9998	<0.0001	4.7	5.5	5.8	101.64	0.053	0.015

R: Correlation Coefficient; RSD: Relative Standard Deviation; RE: Relative Error; LOD: Limit of Detection

Table 3: Brain pharmacokinetic profile of HDD-PIP-NPs (56mg/kg) after single oral administration.

Parameters	Cortex	Cerebellum	Hippocampi
C_max (µg/ml)	7.01±0.97**	4.76±0.24	3.75±0.50
T_max (min)	120±0.00	120±0.00	120±0.00
AUC _0-360 (μg min/ml)	1304.14±115.46**	867.81±47.96	774.67±77.67
AUC _0-∞ (μg min/ml)	1714.20±152.72**	1177.21±102.73	1085.73±36.87
t_1/2(min)	137.53±18.83	143.12±10.77	162.94±22.36

Data are presented as mean±S.D. (n= 4). **p<0.01

Table 4: Physiological parameters of animals subjecting to middle cerebral artery occlusion followed by treatment of 14, 28 and 56 mg/kg HDD-PIP-NPs.

Group	CBF (BPU)	pCO₂ (mmHg)	PO₂(mmHg)	Rectal Temperature (℃)
Sham	289.56±20.43	37.82±2.43	134.33±6.54	36.3±0.38
Vehicle	67.56±9.55	42.47±1.33	67.68±4.55	36.8±0.34
HDD-PIP-NPs(14mg/kg)	166.4±12.34	39.55±2.34	89.33±6.54	36.7±0.23
HDD-PIP-NPs(28mg/kg)	189.3±11.23	38.98±3.02	115.32±7.52	36.7±0.17
HDD-PIP-NPs(56mg/kg)	201.23±15.33	38.55±2.13	128.42±8.54	36.9±0.19

rCBF: regional Cerebral blood flow, BPU: Blood perfusion unit; PCO₂:Partial pressure of carbon dioxide and PO₂: Partial pressure of oxygen.

* All parameters were calculated by using mean value at each time interval.

No competing interests reported.

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Neuroprotective Potential of 6-O-(3-Hexadecyloxy-2-Hydroxypropyl)-Piperine-Nanoparticles in a Rat Model of Cerebral Ischemia-Reperfusion Injury

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Abstract

Purpose

Methods

Results

Conclusion

Figures

1. Background

2. Methods

2.1. Drugs and chemicals

2.2. Synthesis of HDD-PIP-NPs

2.3. Characterizing the HDD-PIP-NPs

2.3.1. Percent yield

2.3.2. Particle size measurement

2.3.3. Drug loading and entrapment efficiency

2.3.4. Surface morphology of nanoparticles

2.4. PIP Release study from HDD-PIP-NPs

2.5. Preparation of Piperine Test Formulations

2.6. Chromatographic system

2.7. Preparation of Standard Solutions and Quality Control (QC) Samples

2.8. Pharmacokinetic Study

2.9. Brain Distribution Study

2.10. Plasma and Brain Sample Preparation

2.11. Experimental Animals Grouping

2.12. Surgical procedure for Transient Focal Cerebral Ischemia Reperfusion (TFCIR)

2.13. Brain Infarction, Brain water content and Neurological score analysis

3. Results

4. Discussion

5. Conclusions

Abbreviations

Declarations

Funding

Author Contribution

Acknowledgement

Data Availability

References

Tables

Additional Declarations

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