Development of Tinidazole Loaded Polymeric Nanoparticles Formulation and its Characterization

Introduction: The development of safe drug delivery systems for a therapeutic agent with less side effects and more bioavailability to the targeted site is very vital in drugs formulation. Tinidazole (TZ) is a drug used to treat giardiasis, amebiasis for colon infections and other infections also such as trichomoniasis, bacterial vaginosis. But the oral bioavailability for the current using drugs low. So, the current study was aimed to develop colon targeted drug delivery system for Tinidazole (TZ) with polymeric nanoparticles (NPs). Methodology: The nanoparticles formulations of TZ were prepared with modified ionic gelation method using chitosan and hydroxypropyl methylcellulose phthalate (HPMCP) are in different combinations by magnetic stirring method followed by temperature modulated solidification. The solvent evaporation method applied to coat TZ nanoparticles with Eudragit S100. The prepared TZ nanoparticle were studied to evaluate physiochemical properties, In-vitro drug release, mucopenetration and In-vivo mucoadhesive studies were carried out. Results: The results of study indicate, 1:1 ratio of chitosan and HPMCP formulation of Original Research Article Devi and Venkateswarlu; JPRI, 33(18): 72-83, 2021; Article no.JPRI.66955 73 nanoparticles provides better spatial interaction between them and TZ with spherical porous and the particles size was diverging between 202 236 nm. In vitro release of TZ followed Higuchi and first order equations better than zero order equation. The drug release results of nanoparticles formulations of TZ indicate that the NPs have potential as a drug delivery system compare to uncoated TZ and coated nanoparticles have comparatively less mucoadhesive detachment force. Conclusion: In conclusion, the study was an evidence to use nanoparticles in colon targeted drug delivery systems for better bioavailability of drugs at targeted site and the biodistribution properties of drugs using nanoparticle will be depend on their composition, particle size and their adhesive abilities.


INTRODUCTION
The emerging of new diseases in recent times around the world are demanding to develop new drug therapeutics to control [1,2]. The usage of current drugs also giving doubt about their efficacy and are becoming resistance towards different diseases, failing in treat them and are may cause for the emerging of new resistant organisms [3,4]. The available drugs around the world have different route of administration i.e., parentals and enteral for treatment and their efficacy dependent on their quantity (Bioavailability) to reach the targeted site after administration [5][6][7]. The bioavailability of drugs depends on drugs' physiochemical and biological properties and the different ingredients used in their formulations for safe drug delivery system to targeted sites [5,8,9]. So, the development of safe drug delivery systems for a therapeutic agent with less side effects and more bioavailability to the targeted site is very vital in their formulation [10,11]. In order to overcome this situation innovative drug delivery systems are developing through colloidal drug carriers such as nanoparticles, microspheres, liposomes, polymer micelles [12]. These molecules work as carriers for new therapeutic drugs in formulations in the achievement of more bioavailability at targeted sites for their intended use. With development of this nanotechnology, development of targeted drug delivery system obtained more desirability in growth of new formulations to current drugs and new drugs [13,14].
The colon infections are one of the major diseases in the current world people [15]. There were different colon infections; colitis can be caused by viruses, bacteria, and parasites, Inflammatory bowel disease, Ischemic colitis, Allergic reactions and Drug-induced colitis [16][17][18]. These infections are directly or indirectly causing the mortality [15]. Different medications were available to treat the colon infection depends on infections' symptoms [19][20][21].
is one of such drugs used to treat giardiasis, amebiasis for colon infections and other infections also such as trichomoniasis, bacterial vaginosis [22,23]. Current marketed preparations of tinidazole are conventional immediate release tablets and thus do not provide localized effect in colon. Thus, there is a need to develop colon specific oral drug delivery systems for Tinidazole with minimum drug release before it reaches the colon and immediate release as soon as it reaches the colon.

Chemical and Drugs
Tinidazole was obtained as free sample from M/s Albert David Ltd., Ghaziabad, India. Chitosan was procured from Yarrow Chem Products, Mumbai, India. Hydroxypropyl Methylcellulose (HPMCP) was obtained from Amrutha Organics, Hyderabad, India. The other chemicals and solvents used in current research are analytical grade.

Drug polymer Compatibility Studies using Fourier Transform Infrared Spectroscopy (FTIR)
FTIR absorption spectra of pure drug, all the polymers used, and the combination of drug and polymers were taken to confirm the identity of the drug and to detect the interaction of the drug with the excipients [24].

Thermal Properties by Differential Scanning Calorimetry (DSC)
Thermal properties of pure drug, optimized formulations and stability studies subjected samples of optimized formulations were evaluated by Differential scanning calorimetry (DSC) using a Diamond DSC (Mettler Star SW 8.10). The analysis was performed from 500 C to 2000 C temperature range at a rate of 5 0 C min-1 under nitrogen flow of 25 ml min-1 [25,26].

Preparation of Nanoparticles (NPs)
The ionic gelation method with magnetic stirring at room temp was used in preparation of NPs formulation with TZ (0.05-0.1% w/v) was carried to formulate with different combinations of chitosan (0.1-0.2% w/v) in acetic acid (1%v/v pH5.0), which is one of best polysaccharide in the current world with antibiotic activity, hemostatic agent and improves the drug delivery and HPMCP (0.1-0.2%w/v) in 0.1M sodium hydroxide solution (Table 1) which is an cellulose derivative protect the drug from degradation by gastric acids. The completed dispersion was centrifuged at 20000 rpm for 30minutes at 4°C. The supernatant was collected for estimation of TZ and the pellet (bottom material) was washed with double distilled water lyophilized using trehalose dihydrate as a cryoprotectant at -55º C at a pressure of 0.01 mm of Hg. Finally optimized that 1:1 ration of chitosan and HPMCP is most suitable for nanoparticles formulation.

Nanoparticles Coating
Acetone solution 12% w/v was used to prepare coating solution to coat TZ nanoparticles (dispersed in 1:10) with Eudragit S100 using rota evaporator.

Nanoparticles Dispersions' Lyophiliza -tion (Freeze Drying)
The NPs dispersion was lyophilized to get dry powder using lyophilizer (-49°C and vacuum maintained at 0.120 mBar) and during lyophilization cryoprotectant anhydrous trehalose equivalent to the amount of chitosan was added to the samples. The samples were stored in desiccator for further study.

Evaluation of Nanoparticles
The developed NPs formulation was evaluated by different parameters.

Morphology of NPs
Transmission electron microscopy (Hitachi HD-2300) in the phase contrast mode to visualize the internal matrix and shape of individual nanoparticles.

Thermal analysis of NPs
A differential scanning calorimeter (PerkinElmer Diamond DSC, CT, USA) equipped with an intercooler 1P was used to analyze the thermal behavior of NP samples and physical state of the drug in NPs [27]. The thermograms were recorded over a temperature range of 10-350°C at a heating rate of 10°C/min under nitrogen purge gas maintained at a flow rate of 20 ml/min. The thermograms were analysed using Pyris Manager (v 1.3) software.

Droplet size determination of NPs
The droplet size of the resultant nanosuspension was measured in a dynamic light scattering instrument (Nicomp 380 ZLS, Particle Sizing Systems, CA) by placing the sample in the path of a Helium Neon laser of wavelength 658 nm at a scattering angle of 90° and a temperature of 23°C.

Zeta potential determination of NPs
Dynamic Light Scattering (DLS) Instrument was used to measure Zeta Potential in the electrophoretic light scatting mode (ELS) by scattering angle at -15.06 and a temperature of 23˚C.

Drug encapsulation efficiency
Drug encapsulation efficiency studies of NPs using UV-Visible spectrophotometer [27]. It was calculated by subtracting the amount of TZ in supernatant of formulated NPs with TZ after centrifuge in a 100 kDa Amicon Ultra-15 Centrifugal Filter Unit at 7830 RPM for 30 min at 15°C against suitably diluted filtrate of blank NP dispersion at absorbance 265nm from amount originally added to formulation.

In vitro drug release studies from NPs
In vitro release profiles of TZ from the lyophilized NPs were obtained by a dissolution test in phosphate buffer solution. A regenerated cellulose membrane (44mm) was loaded with lyophilized TZ NPs and place in 100ml of phosphate buffer maintained at 37°C with 100rpm. The 4mL of free medium was collected and replaced with fresh phosphate buffer. The collected solution was quantified for TZ against samples prepared as above with free NPs as blank by UV spectrophotometry at 242nm and cumulative release of TZ was calculated based on a pre-generated the calibration curve. The mechanism of drug release from the NP's was measured by treating the drug dissolution data with the following different release kinetic models., Zero order release (Cumulative percent drug released Vs time) equation, Higuchi's (Cumulative percent drug released Vs square root of time) equation and Korsmeyer and Peppas (Log cumulative percent drug released versus log time) equation [28].

In vivo mucopenetration study
The study was carried out using freshly isolated pig intestinal ileum and is maintained at ice-cold oxygenated phosphate buffer saline (PBS). The mucus sample equilibrated for 20minutes at 37°Cin a vibrator to get homogenous dispersion mucus. The mucus was placed in donor chamber of Franz-diffusion cell having dialysis membrane between receptor and donor chambers for support the mucus. Then 2 ml of TZ loaded NPs were added on the surface of mucus and receptor chamber was filled with PBS. Later, at different time 1itervals 1ml of sample was withdrawn and replaced with equal volume of PBS, conc. Of TZ was determined spectrophotometrically at 277 nm.

Accelerated stability studies
A stability study was carried out on the optimized batch (CHP5) to assess the stability of nanoparticles by placing in stability chamber, adjusted at different temperature, i.e., 40 ± 0.5º C, at a relative humidity (RH) of 75 ± 5%, as well as at 25 ± 2º C and RH of 60 ± 0.5% for a period of 12 weeks by evaluated for physical appearance and drug content.

Drug Polymer Compatibility Studies by Fourier Transform Infrared Spectroscopy (FTIR)
The IR spectrum of pure Tinidazole is characterized by principal absorption peaks at 3405.20 cm-1 and 3332.

Thermal Properties by Differential Scanning Calorimetry (DSC)
The melting range of TZ was observed between 157 -161°C with capillary method and was comparable with standard range of 158-162° C and with DSC it was found to be 160.55° C, the observed values comply with the standard value given in the official monograph (BP), which confirmed the identity and purity of the drug. The observed λmax was 277 nm compared to the standard λmax of 278 (Fig. 3).

Preparation of Nanoparticles
Various factors such as polymer weight ratios, polymer mixing ratios, pH of polymers, effect of sonication time during mixing influences the ionic interaction. Chitosan (0.1-0.2%), HPMCP (0.1-0.2%) and TZ (0.05-0.1%) concentration selected for further studies and it was optimized that 1:1 ration of chitosan and HPMCP is most suitable for nanoparticles formulation.
pH range of chitosan between pH 2 -7 and HPMCP between pH 2-7 has been selected and it was found that 5.5 pH was most suitable for nanoparticles formulation between chitosan and HPMCP. Particle size was increased when preparation pH increased further. The effect of mixing ratio between chitosan and HPMCP during formulation of nanoparticles, the different volumes of HPMCP was added to the chitosan which was kept constant. The minimum particle size was observed with chitosan: HPMCP mixing volume ratio of 1:1 (Table 2).

Size Determination of NPs
The formulated polymeric nanoparticles are spherical in shape and nanometre size range. The recovery of NPs was found to be 89%. The particle size is a critical parameter for evaluation during and after formulation of NPs. The particle size data of NPs dispersions prepared by modified ionic gelation process by using chitosan as polymer with HPMCP was given in Table 2. A few particles with higher particle size were sometime observed when ionic gelation process is not performed with uniform stirring process. At the concentration of chitosan 0.1% w/v the particles size was diverging between 202-236nm and at 0.2% w/v concentration 272-344 nm (Figs. 4 and 5).

Zeta Potential Determination of NPs
The zeta potential is mainly affected by chitosan concentration and varied from 26.9 -49.7 mV when chitosan concentration ranges from 0.1-0.2% w/v (Fig. 6). It may be said that availability of protonated amino groups is higher with increasing concentration of chitosan, and for optimized formulation Zeta potential was found to be 26.9 ± 2.4Mv and its value >25 mV can be indicating nanoparticles stability. In current study zeta potential remained above + .25 mV at the selected pH, thus confirming that the system remains stable and there was no aggregation (Table 3).

Drug Encapsulation Efficiency Studies
An indirect method was used to determine the encapsulation efficiency of NPs prepared via low temperature solidification process. The NP dispersion was centrifuged in a 100 kDa Amicon Ultra-15 Centrifugal Filter Unit and the free drug present in the filtrate was analyzed by UV-Visible spectrophotometer ( Table 3). The percent drug entrapment of the nanoparticles ranged from 28.5-86.7% and was highest at the highest concentration of chitosan and HPMCP. Drug entrapment showed a linear relationship with polymer concentration.

In vitro Drug Release Studies from NPs
In vitro release performed on coated (Eu-CS-HPMCP) and uncoated (CS-HPMCP) nanoparticles to estimate drug release at colonic pH. In the current study, release determined in simulated gastric (0.1M HCl solution) and colonic fluid (pH 6.8), TZ released from the coated nanoparticles after 2 h in 0.1M HCl was 5.57±1.34% whereas in uncoated nanoparticles, it was 16.08 ±5.51% (Table 4).
In vitro drug release from redispersed lyophilized NPs was performed in phosphate buffer pH 7.4 by using a dialysis bag. The samples were analyses at 242nm. The TZ release from the NP's was slow and spread over more than 70 h. The slow release indicated that the drug was in the core surrounded by drug free lipid shell. Data of the in vitro release was fit into different equations and kinetic models to explain the release kinetics of TZ from NP's. The kinetic models used were zero order, first order, higuchi, Hixson Crowell and korsemeyer-peppas models ( Table 5, Figs. 7 A and B).
The bioadhesion detachment force studies on optimized nanoparticles (n=3) showed detachment force up to 12.34x103 dynes/cm2 for coated and for the uncoated it was up to 15.98x103 dynes/cm2. Coated nanoparticles have comparatively less mucoadhesive detachment force, which may be due to decrease in surface amino groups. This reduced mucoadhesion can facilitate in infiltration of nanoparticles to gastric mucosa enhanced penetration and accumulation at the site of infection beneath mucosa.

In-vivo Mucopenetration Studies
FITC labelled Pegylated chitosan nanoparticles showed internalization of the particles more at 5h and 8h. This may be due to reduction of surface charge which accelerates the adhesion and penetration of the NPs in to the mucus. Study conducted at 12h and 24h showed good number of fluorescent particles in the inner mucus layer (Fig. 8).

Accelerated Stability Studies
The optimized formulation (TZ5) of nanoparticles was subjected to two different temperature and humidity conditions for 12 weeks exhibited no change in colour and appearance. The chemical stability results have shown that the percent drug remaining was found to be 99.35 ± 1.12% and 86.45 ± 1.23%, at 25º C and 40º C respectively. There was statistically insignificant difference in bio-adhesion strength of nanoparticles during 12 weeks. The regression analysis of stability data indicates that the drug degradation follows first order kinetics (Table 5). This could be explained by taking surface charge into consideration irrespective of the particle size. There may be less electrostatic interaction between mucin fibre with pegylated nanoparticles which accelerates the process of penetration into the outer mucus and then into the inner mucus, close proximity to the epithelial cells [29]. Also, particles were not cleared with mucus and remained at the site even after the mucus clearance. Pegylated nanoparticles rapidly transported to the mucus as these particles could able to move in low viscosity channels and do not interact significantly with mucus [32]. Additionally, pegylation technique also improves the stability of nanoparticles in mucus and it is very important as particles must diffuse through a thick mucus layer in order to treat underlying infection. There were earlier some reports on the development of colon targeted drug delivery systems and development of tinidazole oral drug delivery system. Mukund and Chaudhari were developed colon-targeted delivery system, which uses lactulose and Tinidazole shown protections against pH 1.2 and pH 6.8 upto six-hour study with best results at pH5.0 [33]. Krishnaiah and his team reported e in vivo availability of guar gum-based colon-targeted tablets of tinidazole in comparison with immediate release tablets of tinidazole in human volunteers and found e immediate release tablets of tinidazole produced a peak plasma concentration (Cmax of 3239 ± 428 ng/ml) at 1.04 ± 0.32 hr (Tmax), whereas colon-targeted tablets produced peak plasma concentration (Cmax of 2158 ± 78 ng/ml) at 14.9 ± 1.6 hr. The delayed Tmax, decreased Cmax, and Ka, and unaltered bioavailability and elimination half-life of tinidazole from guar gumbased colon-targeted tinidazole tablets [34]. By comparing with other earlier reports and current study was first in development of tinidazole loaded NPs with chitosan and results confirm the novel concept of mucopenetration by decreasing or shielding the cationic charge on polymers like chitosan by modifying the surface chemistry of such polymers in order to improve the motility of nanoparticles in mucus membrane.

CONCLUSION
The current study was aimed to develop Tinidazole NPs using magnetic stirring method followed by temperature modulated solidification and successfully developed TZ loaded nanoparticle with NPs of low nm size using lipid chitosan and nontoxic surfactants like eutdragit S100. The developed TZ loaded NPs were evaluated by different physiochemical parameters and were produced efficient results and also In vitro release of TZ followed Higuchi and first order equations better than zero order equation. The current study results for TZ NPs indicate that the NPs have potential as a drug delivery system. Furthermore, they may have utility for site specific drug delivery since the small size of the particle and its biodistribution properties may allow their delivery to target sites.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.