Chitosan Encapsulated Meloxicam Nanoparticles for Sustained Drug Delivery Applications: Preparation, Characterization, and Pharmacokinetics in Wistar Rats

Meloxicam (MLX) is currently used in the therapeutic management of both acute and chronic inflammatory disorders such as pain, injuries, osteoarthritis, and rheumatoid arthritis in both humans and animals. Gastrointestinal toxicity and occasional renal toxicity were observed in patients taking it for a long-term period. Meloxicam’s late attainment of peak plasma concentration results in a slow onset of action. The goal of the current study was to prepare and characterize chitosan encapsulated meloxicam nanoparticles (CEMNPs) with high bioavailability and less gastro intestinal toxicity in order to prevent such issues. The size of the prepared CEMNPs was approximately 110–220 nm with a zetapotential of +39.9 mV and polydispersity index of 0.268, suggesting that they were uniformly dispersed nanoparticles. The FTIR and UV-Vis spectroscopy have confirmed the presence of MLX in the prepared CEMNPs. The pharmacokinetics have been studied with three groups of male Wistar rats receiving either of the treatments, viz., 4 mg·kg−1 of MLX and 1 or 4 mg·kg−1 of CEMNPs. Plasma samples were collected until 48 h post administration, and concentrations of MLX were quantified by using reverse (C18) phase HPLC. Non-compartmental analysis was applied to determine pharmacokinetic variables. Upon oral administration, the maximum concentration (Cmax) was reached in 4 h for CEMNPs and 6 h for MLX. The mean area under the plasma MLX concentration-time curve from ‘zero’ to infinity (AUC0–∞), half-life (t1/2β), and mean resident time (MRT) of 1 mg·kg−1 of CEMNPs was 1.4-, 2-, and 1.8-fold greater than 4 mg·kg−1 of MLX. The prepared CEMNPs demonstrated quicker absorption and prolonged release along with a significant improvement in the bioavailability of MLX, paving a prospective path for the development of drugs with enhanced bioavailability with less side effects.


Introduction
The expression of pain consequent to inflammation is a necessity, and it favors veterinarians to identify and treat the cause of pain, which will inherently improve the overall welfare of the animal [1]. Part of treating the pain is understanding the mechanisms behind analgesic To increase drug bioavailability or precise targeted distribution at the site of action, polymeric nanoparticles stand out as a crucial instrument. Polymers have the potential to be suitable for meeting the criteria of each unique medication delivery system due to their versatility [26]. In general, polymeric nanoparticles can be optimized to improve the drug bioavailability, either by enhancing their solubility or enabling their transit through the biologic membranes to increase their absorption [27].
Chitosan, a naturally occurring hydrophilic cationic polysaccharide obtained from crustacean shells and fungal cell walls comprised of randomly dispersed β-1-4 linked D-glucosamine and N-acetylglucosamine, is one of the extensively used polymers for the synthesis of new drug delivery systems. Recently, the US FDA approved chitosan for the delivery of nanoparticles [28]. Due to its nontoxic nature, bio-adhesive property, biocompatibility, biodegradable property, and target specificity, chitosan can be used to prepare nanoparticles for drug delivery [29][30][31]. Chitosan-based nanomaterials exhibit superior drug absorption due to their GI luminal protection, mucoadhesive nature, permeability enhancement, controlled drug release, and efflux inhibition [30]. In recent times, few attempts have been made to synthesize MLX nanoparticles for effective delivery [17,18,32,33]. After a thorough review of the literature, and with the available information regarding chitosan-encapsulated meloxicam nanoparticles, the current investigation was carried out to explore the beneficial effects of chitosan-encapsulated meloxicam nanoparticles with respect to their pharmacokinetic behavior in male Wistar rats.

Preparation and Characterization of CEMNPs
In the present study, the CEMNPs were prepared by the ionic gelation crosslinking process. Due to the protonation of the amine group of chitosan in acidic pH, it interacts with polyanion TPP. The instantaneous formation of CEMNPs were observed upon adding the MLX dissolved in a methanol and TPP solution mixture to the chitosan solution. Because of the interaction between the negatively charged TPP and MLX, as MLX exists as an anion in neutral or weakly basic solution [34] with positively charged chitosan during ionic gelation, this resulted in the formation of CEMNPs.
The transmission electron microscopic (TEM) and scanning electron microscopic (SEM) micrographs of the CEMNPs are shown in Figures 1a and 1b, respectively. The electron microscopic analysis revealed that the shape of the CEMNPs was round to cuboidal in shape, with a size distribution of 110-200 nm. The energy-dispersive spectroscopy analysis ( Figure 2 and Supplementary Table S1) revealed the presence of C, N, O, and S, which confirmed the presence of MLX in the prepared CEMNPs (Chitosan-C 12 H 24 N 2 O 9 ; MLX-C 14      The DLS technique is considered a sensitive and valuable method for measurement of NPs. This method yields a particle's hydrodynamic diameter [35]. The hydrodynamic diameter of the CEMNPs was measured in 0.1% carboxymethyl cellulose (CMC), which yielded 138.5 nm (Figure 3a). The previous studies with chitosan nanoparticles reported the size range of 90-690 nm [33,[36][37][38].
The uniformity of particles in the solution was estimated using the polydispersity index value. A larger size distribution in the particle sample is indicated by higher polydispersity index values. The range of the polydispersity index is between '0′ and '1′, with '0′ denoting monodisperse and '1′ denoting polydisperse [39]. The polydispersity index of the CEMNPs measured in 0.1% CMC was 0.268 (Figure 3a), indicating that the prepared nanoparticles were monodispersed.
The zeta potential is a physical property exhibited by any particle in suspension. It depends on the surface charge and is important for the stability of NPs in suspension. According to conventional wisdom, charged particles with zeta potentials of above +30 mV or below −30 mV exhibit adequate electrostatic repulsion to provide colloidal stability [40]. In the present study, the prepared CEMNPs were positively charged with a zeta potential of +39.9 mV (Figure 3b). Due to the presence of free amine groups on the polymer, the positive zeta potential value of CEMNPs shows that the nanoparticle surface has positively charged [41,42]. In the previous studies, the zeta potential for chitosan nanoparticles was reported as positively charged [33,[36][37][38]. The DLS technique is considered a sensitive and valuable method for measurement of NPs. This method yields a particle's hydrodynamic diameter [35]. The hydrodynamic diameter of the CEMNPs was measured in 0.1% carboxymethyl cellulose (CMC), which yielded 138.5 nm (Figure 3a). The previous studies with chitosan nanoparticles reported the size range of 90-690 nm [33,[36][37][38]. The uniformity of particles in the solution was estimated using the polydispersity index value. A larger size distribution in the particle sample is indicated by higher polydispersity index values. The range of the polydispersity index is between '0' and '1', with '0' denoting monodisperse and '1' denoting polydisperse [39]. The polydispersity index of the CEMNPs measured in 0.1% CMC was 0.268 (Figure 3a), indicating that the prepared nanoparticles were monodispersed.
The zeta potential is a physical property exhibited by any particle in suspension. It depends on the surface charge and is important for the stability of NPs in suspension. According to conventional wisdom, charged particles with zeta potentials of above +30 mV or below −30 mV exhibit adequate electrostatic repulsion to provide colloidal stability [40]. In the present study, the prepared CEMNPs were positively charged with a zeta potential of +39.9 mV (Figure 3b). Due to the presence of free amine groups on the polymer, the positive zeta potential value of CEMNPs shows that the nanoparticle surface has positively charged [41,42]. In the previous studies, the zeta potential for chitosan nanoparticles was reported as positively charged [33,[36][37][38].
The UV spectral analysis of MLX and the CEMNPs was obtained by carrying the scanning over the wavelength range of 200-400 nm, and the resulting spectrum is depicted in Figure 4. The primary peak was found to be around 359 nm. The prepared CEMNPs showed similar peaks with less intensity, which confirmed the presence of MLX. These results are in good agreement with the previous reports [43][44][45][46][47]. The UV spectral analysis of MLX and the CEMNPs was obtained by carrying the scanning over the wavelength range of 200-400 nm, and the resulting spectrum is depicted in Figure 4. The primary peak was found to be around 359 nm. The prepared CEMNPs showed similar peaks with less intensity, which confirmed the presence of MLX. These results are in good agreement with the previous reports [43][44][45][46][47].

Encapsulation Efficiency (%)
The encapsulation efficiency (%) as analyzed by HPLC in the supernatant devoid of CEMNPs was found to be 96.49% for the CEMNPs and was in good agreement with Mohammed et al. [23]. The high drug entrapment efficiency of the CEMNPs could be explained by an efficient ionic interaction between MLX, which is a weak acid, with two pKa values (pKa l = 1.09, pKa 2 = 4.18) and chitosan. During the preparation of the CEMNPs, a mixture of MLX and TPP solution was slowly added dropwise to the chitosan solution, which allowed the interaction of the negatively charged MLX and TPP with positively charged amine groups in the chitosan, resulting in the formation of high MLX entrapment [28,48]. and 1039.49 cm −1 (S=O stretching vibrations of organic sulfoxide). The results from the FTIR of the CEMNPs showed the existence of prominent peaks of MLX, which indicates there is no interaction occurring between MLX and the polymer during the preparation of nanoparticles.

Encapsulation Efficiency (%)
The encapsulation efficiency (%) as analyzed by HPLC in the supernatant devoid of CEMNPs was found to be 96.49% for the CEMNPs and was in good agreement with Mohammed et al. [23]. The high drug entrapment efficiency of the CEMNPs could be explained by an efficient ionic interaction between MLX, which is a weak acid, with two pKa values (pKa l = 1.09, pKa 2 = 4.18) and chitosan. During the preparation of the CEMNPs, a mixture of MLX and TPP solution was slowly added dropwise to the chitosan solution, which allowed the interaction of the negatively charged MLX and TPP with positively charged amine groups in the chitosan, resulting in the formation of high MLX entrapment [28,48].

In Vitro Release Kinetics
The in vitro cumulative drug release of MLX from the CEMNPs over a period of 48 h at pH 4.8 and 7.4 is depicted in Figure 6. According to the release curves, MLX released from the CEMNPs over a 48 h period was slower at pH 7.4 as compared to pH 4.8. A total amount of 79.36% and 55.86% of loaded MLX was released from the CEMNPs at pH 4.8 and 7.4, respectively, within 48 h. When compared to pH 7.4, the release of MLX from the CEMNPs was more rapid at acidic pH. This might be due to the expanded swelling property of chitosan at acidic pH. Since chitosan is more soluble and degradable in acidic pH than neutral and/or alkaline pH, it is possible that the delayed release of MLX from the CEMNPs at pH 7.4 is caused by the nature of this molecule [49,50]. The controlled release of MLX was observed from the CEMNPs without any initial burst, which infers that the encapsulation of the drug was homogenous. The lack of the initial burst suggests that the drug was properly encapsulated in the nanoparticles, and there was no surface deposition [51,52]. The ability of chitosan to hydrate when in contact with dissolution media results in the formation of gelatinous mass, which acts as a retardant material for the drug to diffuse out, which helps in sustained drug release [53].

In Vitro Release Kinetics
The in vitro cumulative drug release of MLX from the CEMNPs over a period of 48 h at pH 4.8 and 7.4 is depicted in Figure 6. According to the release curves, MLX released from the CEMNPs over a 48 h period was slower at pH 7.4 as compared to pH 4.8. A total amount of 79.36% and 55.86% of loaded MLX was released from the CEMNPs at pH 4.8 and 7.4, respectively, within 48 h. When compared to pH 7.4, the release of MLX from the CEMNPs was more rapid at acidic pH. This might be due to the expanded swelling property of chitosan at acidic pH. Since chitosan is more soluble and degradable in acidic pH than neutral and/or alkaline pH, it is possible that the delayed release of MLX from the CEMNPs at pH 7.4 is caused by the nature of this molecule [49,50]. The controlled release of MLX was observed from the CEMNPs without any initial burst, which infers that the encapsulation of the drug was homogenous. The lack of the initial burst suggests that the drug was properly encapsulated in the nanoparticles, and there was no surface deposition [51,52]. The ability of chitosan to hydrate when in contact with dissolution media results in the formation of gelatinous mass, which acts as a retardant material for the drug to diffuse out, which helps in sustained drug release [53]. The MLX release data from the CEMNPs dispersion was subjected to kinetic analysis using several kinetic models, and the results are presented in Table 1. The kinetic model with the best computed correlation coefficient (R 2 ) was chosen as the MLX release mech-  The MLX release data from the CEMNPs dispersion was subjected to kinetic analysis using several kinetic models, and the results are presented in Table 1. The kinetic model with the best computed correlation coefficient (R 2 ) was chosen as the MLX release mechanism. The release data of MLX from the CEMNPs dispersion was better explained by the Baker-Lonsdale models at both pH points, which indicates that the drug release was happening from the matrix type of formulation and nano capsules. According to the release exponent 'n', calculated using the Korsmeyer-Peppas equation, the drug's diffusion from the CEMNPs was anomalous and non-Fickian, with values of 0.66 and 0.67 at pH 4.8 and 7.4, respectively. These results are consistent with the earlier published kinetic investigations of MLX release data from chitosan nanoparticles [28].
The mean apparent volume of the distribution of MLX was 0.265 ± 0.006 L.kg −1 , 0.088 ± 0.016 and 0.183 ± 0.016 for 4 mg·kg −1 of MLX, and 1 or 4 mg·kg −1 of CEMNPs, respectively, following oral administration. This indicates that the apparent volume of distribution is substantially higher for plain MLX as compared to CEMNPs. Thus, CEMNPs may have better target efficiency.
The mean area under the plasma MLX concentration-time curve from zero to infinity (AUC 0-∞ ), half-life, and mean resident time of 1 mg·kg −1 of CEMNPs was 1.4-, 2-, and 1.8-fold greater than 4 mg·kg −1 of MLX standard drug. The possible reason for the abovementioned results is mainly due to the protective effect of the chitosan. This is because the encapsulated MLX needs to be released from the CEMNPs into circulation and then distributed to tissues and eliminated [37].
Meloxicam is an enolic acidic NSAID drug with anti-inflammatory, analgesic, and antipyretic activities [8,9]. The aqueous thermodynamic solubility and dissolution rate of MLX is poor to low. The low molecular weight of 351.4 kDa helps to maintain the well permeability. However, MLX has an 89% bioavailability after dissolution, and its poor dissolving property restricts both its absorption rate and onset of action [17].
In the present study, we have prepared MLX nanoparticles with chitosan to improve absorption rate with less GI side effects. The features of chitosan-based nanomaterials, such as the protection of pharmaceuticals against gastrointestinal degradation, higher mucoadhesion, improved permeability, controlled drug release, and efflux pump suppression, have enhanced the fast and sustained drug delivery [29,30,[54][55][56].
Kürti et al. [17] have reported similar kind of results with MLX-polyvinylpyrrolidone nanoparticle pharmacokinetics in male Sprague-Dawley rats. The C max and AUC were increased 2.4-and 2-fold, respectively, when MLX-polyvinylpyrrolidone nanoparticles were administered orally to rats, as compared to plain MLX.
The results of this study were in similar to Nagai et al. [18], who designed and studied the pharmacokinetics of MLX solid nanoparticles in male Dark Agouti rats. The time taken to reach the maximum concentration (T max ) of MLX solid nanoparticles was shorter than that of plain MLX, and the intestinal penetration of MLX-NPs was significantly higher in comparison with plain MLX. The area under the plasma MLX concentration-time curve for MLX-NPs was 5-fold higher than that for plain MLX, and the AUC in rats administered 0.05 mg/kg MLX-NPs were similar to rats administered the therapeutic dose of 0.2 mg/kg plain MLX.
The prolonged MRT of MLX in the present study was in accordance with the reports of Wang et al. [57], who studied the in vivo pharmacokinetics of hybrid thermosensitive MLX chitosan gel in Sprague-Dawley rats. The MLX chitosan-glycerol gel significantly prolonged the MLX elimination time by 24 h, and a 1.8-fold increase in ACU was observed compared to plain MLX.

Preparation of Chitosan-Encapsulated Meloxicam Nanoparticles (CEMNPs)
Ionic gelation was used for the preparation of chitosan-encapsulated meloxicam nanoparticles (CEMNPs) with minor modification in the method described by Duse·et al. [58]. In a nutshell, 1 mg·mL −1 of chitosan was dissolved in 0.5% acetic acid solution and stirred continuously at 120 rpm over the course of the night at room temperature. An aqueous solution of sodium tripolyphosphate (NaTPP) at a concentration of 1 mg·mL −1 was prepared. Meloxicam was dissolved in methanol at a concentration of 1 mg·mL −1 solution, and desired volume was mixed with NaTPP solution. The CEMNPs were prepared by stirring the chitosan solution at 600 rpm using a magnetic stirrer, and dropwise meloxicam-NaTPP solution was added at the ratio of 3:1 (Chitosan:NaTPP). After being brought to a pH of 5.5, the obtained nanoparticle suspension was agitated for 30 min and then centrifuged at 16,000 rpm for 30 min at 14 • C. The supernatant was removed, and the wet pellet of CEM-NPs was collected and washed with ascending grade ethanol. Then, they were lyophilized with freeze-dryer (ScanLaf, CoolsafeFreezedryer, LaboGene; LillerØd, Denmark), and the powder form CEMNPs was stored at 4 • C for further analysis.

Transmission Electron Microscopic (TEM) and Scanning Electron Microscopic (SEM) with Energy Dispersive Spectroscopy (EDS) Analysis
The TEM analysis was conducted using JOEL 30,100 TEM machine to determine the size and shape of TiO 2 NPs. Thin films of the sample were prepared on a TEM grid by just dropping a very small amount of the sample on the grid, extra sample was removed, and then the film on the TEM grid was allowed to dry by putting it under a mercury lamp for 5 min.
The SEM analysis was conducted using CARL-ZEISS AG-ULTRA 55 machine equipped with EDS analyzer to determine the size, shape, and elemental distribution of CEMNPs. Thin films of the sample were prepared on a SEM grid by just dropping a very small amount of the sample on the grid, extra sample was removed, and then the film on the SEM grid was allowed to gold sputter (EMITECHK-550X; Ashford, UK) for 1 min before taking the images and EDS analysis.
The instrument was equipped with 10 mW or 100 mW laser beam at a wavelength of 532 nm. Surface charge of CEMNPs was measured by an electrophoretic light scattering technique using a fold capillary cuvette. Zeta potential was measured with an electrode voltage of 3.9 V at 25 • C.

UV-Visible Spectra Analysis
The UV-VIS spectrophotometer (UV-2450, Shimadzu, Kyoto, Japan) was employed to assess the MLX and CEMNPs' UV-visible spectrum after diluting a tiny amount of the sample in distilled water and scanning for absorption maxima.

Fourier Transform Infrared (FTIR) Spectroscopy
FTIR spectral analysis of MLX and CEMNPs were performed using a FT/IR 6600 FTIR spectrometer (Jasco Corporation, Tokyo, Japan) over a range from 4500 to 350 cm −1 . Potassium bromide was used for the preparation of respected sample compressed discs. The number of scans used was 16 at a resolution of 4 cm −1 with a scanning speed of 2 mm per second. FTIR was used to evaluate the types of functional groups present in the prepared nanoparticles and will confirm the presence of drug after successful encapsulation.

Encapsulation Efficiency
The percent encapsulation efficiency (EE) was evaluated by the method described by Chuah et al. [59] with slight modifications. The prepared CEMNPs were centrifuged at 12,000 rpm for 30 min. The supernatant containing unbound MLX was separated, and the concentration was determined by using a validated reverse phase (C 18 ) high-performance liquid chromatography (HPLC; Shimadzu ® , Japan) assay [54]. The EE% was calculated using the following Equation (1): (Total amount of meloxicam − amount of free meloxicam) Total amount of meloxicam × 100 (1)

In Vitro Release Kinetic Studies
The drug release kinetics (in vitro) were carried out by using the dialysis membrane as reported by Rajashekaraiah et al. [38], with minor modifications. The dialysis bag technique entails dispersing 3 mg of the CEMNPs in 1 mL of the release medium and placing in a presoaked dialysis bag (12,000-14,000 MWCO, M/s. Sigma-Aldrich, St. Louis, MO, USA, surface area of 20.5 cm 2 ), which was then submerged in a beaker that contained 50 mL of the test media maintained at 37 ± 0.5 • C in a temperature-controlled shaking water bath at 50 rpm, maintained for 48 h. The release media were phosphate buffer solution (PBS) of pH 7.4 and acetate buffer of pH 4.8. At predetermined time intervals (0.5, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 24.0, and 48.0 h), 1 mL aliquots were collected and replaced with the same volume of fresh media to maintain sink conditions. The MLX concentration in release media was determined using HPLC [60]. Each sample has triplicate copies of the analytical run. The intensity of absorption was plotted against time, which gave the desorption profile of CEMNPs. The cumulative percent release was calculated for each time point. The data obtained was subjected to windows-driven KineticDS3 software [61] to get the best-fitted in vitro release kinetic model to determine the rate of MLX release from CEMNPs. Different mathematical modeling drug release equations were applied to perform drug release kinetics such as zero-order Equation (2), first-order Equation (3), Korsmeyer-Peppas Equation (4), Higuchi Equation (5), and Baker-Lonsdale Equation (6): where Q t is the cumulative amount of drug release, Q 0 is the initial amount of drug, K 0 is the zero-order release constant, and t is the time.
where C 0 is the initial concentration of drug, K is the first order rate constant, and t is the time.
where M t /M is the cumulative drug release, K is the release constant, t is the time, and n is the release exponent.
where Q is the cumulative drug release, K is Higuchi release constant, and t is the time.
where K is the rate constant, and t is the time.

Instrumentation
The HPLC system (Shimadzu ® , Japan) consisted of an LC-20AD quaternary gradient pump, a rheodyne manual injector with 20 µL loop, and an SPD-20 AV UV-Vis detector. The analytical column employed was reverse phase (RP-C 18 ) column (4.6 mm × 250 mm, 5 µm particle size). The mobile phase comprised 46 parts of solution 'A': acetonitrile and 54 parts of solution 'B': 1% glacial acetic acid, in the ratio of 46:54 (v/v). The mobile phase liquid was filtered through membrane filter (0.22 µm diameter) and later degassed with the help of ultrasonic cleaner (Sonica ® , Soltec Soluzion Technologiche, Basaldella, Italy). The flow rate was kept at 1.0 mL·min −1 with run time of 10 min for each sample. Chromatography was performed at 40 • C with detection at 355 nm using UV detector [60]. The room temperature was maintained at 23 • C during the assay. The concentration of the analyte in the samples was determined from the standard curve constructed for the purpose. The retention time of MLX in the present assay study was 7.248 min.

Standardization and Validation of Assay
A stock solution of MLX standard with a concentration of 100 µg·mL −1 was prepared by dissolving 5 mg of MLX standard in 50 mL of methanol. Working stock solutions with concentrations of 0, 0. 25,1,5,10,15,25,50, and 100 µg·mL −1 were created by diluting the MLX stock standard in methanol. In order to avoid cross-contamination and evaporation, standards were aliquoted into 2 mL amber-colored vials wrapped with foil to block light and stored at −20 • C.
The drug concentration (X-axis) was plotted against the peak area (Y-axis) to create the standard curve. The standard curve had an R 2 value of 0.999, and it was linear in the range of 0.1 to 100 µg·mL −1 . A typical MLX chromatogram and calibration curve were depicted in Figures 8 and 9, respectively. The limit of detection (LOD) and limit of quantification (LOQ) were determined to be 2.8 and 8.5 ng·mL −1 , respectively. The precision and accuracy of the assay were assessed using samples at concentration 1, 10, and 25 µg·mL −1 . The intraday and interday assay coefficient of variation was less than 7.64% (Table 3), and the percent recovery of MLX in rat plasma was 97.24%.

Standardization and Validation of Assay
A stock solution of MLX standard with a concentration of 100 µ g·mL −l was prepared by dissolving 5 mg of MLX standard in 50 mL of methanol. Working stock solutions with concentrations of 0, 0. 25,1,5,10,15,25,50, and 100 µ g·mL −l were created by diluting the MLX stock standard in methanol. In order to avoid cross-contamination and evaporation, standards were aliquoted into 2 mL amber-colored vials wrapped with foil to block light and stored at −20 °C.
The drug concentration (X-axis) was plotted against the peak area (Y-axis) to create the standard curve. The standard curve had an R 2 value of 0.999, and it was linear in the range of 0.1 to 100 µ g·mL −l . A typical MLX chromatogram and calibration curve were depicted in Figures 8 and 9, respectively. The limit of detection (LOD) and limit of quantification (LOQ) were determined to be 2.8 and 8.5 ng.ml −1 , respectively. The precision and accuracy of the assay were assessed using samples at concentration 1, 10, and 25 μg·mL −1 . The intraday and interday assay coefficient of variation was less than 7.64% (Table 3), and the percent recovery of MLX in rat plasma was 97.24%.

Experimental Animals
Male Wistar rats (N = 99) of 8 weeks of age and weighing 175-185 g were procured from the authorized vendor (Biogen ® Laboratory Animal Facility, Bengaluru-562107, KS, India). MLX is metabolized in the liver through cytochrome P450 2C isoenzymes, particularly CYP450 2C11 isoenzyme, which is absent in female rats. This leads to slower elimination of MLX from female rats. In light of these constraints for female rats, male rats  MLX is metabolized in the liver through cytochrome P450 2C isoenzymes, particularly CYP450 2C11 isoenzyme, which is absent in female rats. This leads to slower elimination of MLX from female rats. In light of these constraints for female rats, male rats were used for this study [62]. All the rats were housed in Small Animal Facility of Veterinary College, Bengaluru, in polypropylene cages at an ambient temperature of 23 ± 2 • C and 55 ± 5% of relative humidity, with 12 h:12 h of light and dark cycle. Animals were kept on ad libitum feed and water. After one week of acclimatization, the experimental rats were randomly divided into three groups (n = 33). Freshly prepared drug solutions were suspended in carboxymethyl cellulose (CMC) (0.1% w/v) and subjected to ultrasonication by using an ultrasonic cleaner for 30 min and stirred over vortex agitator for about 5 min in order to obtain homogenized suspension. After overnight fasting, animals were weighed before dosing, and MLX suspension (CMC; 0.1 w/v) was administered through oral gavage as single dose of 4 mg·kg −1 to Group I, CEMNPs @ dose rate equivalent to 1 mg·kg −1 of MLX to Group-II, and CEMNPs @ dose rate equivalent to 4 mg·kg −1 of MLX to Group-III. Institutional animal ethics committee approval was obtained (VCH/IAEC/2021/123, dated 27 July 2021) prior to experimental studies in Wistar rats, and all maneuvers involved in animal studies were according to the guidelines of Committee for the Purpose of Control and Supervision of Experiments in Animals (CPCSEA, New Delhi, India).

Blood Sampling
Blood samples were collected at 0.0, 0.16, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 4.0, 6.0, 12.0, 24.0, and 48.0 h postdosing from experimental rats and transferred to heparinized tube. Multiple numbers of rats were used for serial collection of blood in some time points, with at least 18 h intervals between collections. The blood samples were centrifuged at 5500 rpm, and the resultant plasma was stored at −80 • C until subjected to quantitative analysis of MLX.

Plasma Samples for HPLC Analysis
To 0.1 mL of plasma in 2 mL Eppendorf tubes, 0.55 mL of a 0.1 M sodium dihydrogen phosphate buffer (pH 3.3) was added. Proteins were precipitated by the addition of 50 µL of 70% perchloric acid, and tubes were agitated in a vortex mixer for minimum of one min. To this solution, 50 µL of acetonitrile was added, and the tubes were agitated again for one min. The final solution was centrifuged for 10 min at 4500 rpm. Following centrifugation, a 0.22 µm nylon membrane syringe filter was used to separate the clear supernatant, and 20 µL of this was injected into HPLC system for quantifying MLX [22,60].

Pharmacokinetic (PK) Analysis of Meloxicam
The mean plasma MLX concentration vs. time curve was plotted for all the three groups. The pharmacokinetic parameters were derived by using a menu-driven add-in program for Microsoft Excel written in visual basic (PK Solver; version 2.0) validated previously [63]. After subjecting the data of mean plasma MLX concentration vs. time intervals of different groups to a non-compartment model with no lag time, the pharmacokinetic variables were derived.

Statistical Analysis
The mean plasma MLX concentration and pharmacokinetic data obtained were subjected to one-way ANOVA. Tukey's post hoc multiple comparison test using GraphPad Prism software program (GraphPad ® software Inc., Version 8.4.3; San Diego, CA, USA) was applied to determine the significant difference between the set of data. The difference was considered significant at p < 0.05 or lower.

Conclusions
In conclusion, compared to plain MLX, the CEMNPs have rapid absorption through oral administration, a lengthy half-life, long mean residence time, and comparatively low apparent volume of distribution, and clearance demonstrated the sustained releasing impact of CEMNPs. This pharmacokinetic trait contributed to the explanation of the enhanced drug delivery, rapid onset of action, and extended activity of MLX following a single low oral dose of chitosan-encapsulated meloxicam nanoparticles in experimental rats. This chitosan-based drug-delivery system appears to be promising for sustained drug delivery based on the correlation between the in vitro and in vivo outcomes of this study and thus necessitates the appropriate clinical studies for potential application in therapeutics.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.