Antimicrobial and anti-inflammatory potential of thermoresponsive in situ gel loaded with metronidazole microsponges for localized treatment of periodontitis

Antimicrobial and anti-inflammatory potential of thermoresponsive in situ gel loaded with metronidazole microsponges for localized treatment of periodontitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antimicrobial and anti-inflammatory potential of thermoresponsive in situ gel loaded with metronidazole microsponges for localized treatment of periodontitis Eman S. Abd El-Reheim, Khaled M. A. Hassanein, Helal F. Hetta, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8679532/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Periodontitis is an inflammatory disease that goes deeply into the tissues, causing loss of alveolar bone and supporting connective tissues. Severe periodontal diseases are estimated to affect around 19% of the global adult population, representing more than 1 billion cases worldwide. Metronidazole (MTZ) is frequently used to inhibit anaerobic bacteria in periodontal disease. This study aimed to fabricate MTZ-eudragit microsponges (MSPs) and then loading them into gel to evaluate its sustained local action in the buccal cavity. To optimize MTZ- MSPs, different variables such as drug:polymer ratio, polymer:solvent ratio and stirring rate were studied. characterization of MSPs, such as thermal behavior, surface morphology, particle size and drug release, were studied. The optimized MSPs were spherical with numerous pores on the surface (sponge-like particles). These optimized MSPs also showed high entrapment efficiency (87 ± 1%), mean particle size of 45 ± 1 µm (PDI 0.2 ± 0.1) and sustained drug release (57 ± 1% through 12 hours). The optimized MSPs were then incorporated into a thermoresponsive gel (20% PF-127/2% PF-68/0.5% HPMC). MSPs-containing gel was free flowing at room temperature and gel in the buccal cavity, retarding drug release and enhancing the mucoadhesion and antibacterial activity. studies on rats revealed that this gel is an effective and innovative approach to treating periodontitis. Conclusion, MSPs-loaded gel is a promising vehicle for MTZ to treat periodontitis. Metronidazole Microsponges Periodontal disease In situ gel Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Periodontitis is an inflammatory condition that spreads deeply into the tissues, resulting in the loss of supporting connective tissue and alveolar bone. Periodontitis causes the formation of soft tissue pockets or widened fissures between the gingiva and the tooth root (Page and Eke 2007 ). It is estimated that over 19% of adults globally suffer from severe periodontal disorders, contributing to over 1 billion cases worldwide (Wu, Yuan et al. 2020). The initial therapy for periodontitis is scaling and root planning (SRP), mechanical debridement of surfaces, and guidance on oral hygiene. Apart from conservative measures, antimicrobial agents can be used locally or systemically to treat periodontitis. It is challenging to provide antimicrobial agents systemically, which results in insufficient drug concentration in the periodontal pocket. Furthermore, using large doses of these drugs can have several negative effects. However, using these drugs locally in the pockets of periodontal disease is an advanced approach to suppress or eradicate the pathogenic microorganisms and also to regulate the inflammatory response in the tissues (Suvan, Leira et al. 2020 ). Metronidazole (MTZ) is a frequently used antimicrobial agent to inhibit anaerobic microorganisms in periodontal disease and also to treat several infections such as trichomoniasis, giardiasis, amebiasis, parasitic infections and gastrointestinal infections (Ceruelos, Romero-Quezada et al. 2019 )(Weir and Le 2022). Local periodontal MTZ delivery is very advantageous, both in terms of increasing the concentration directly in the infected site and minimizing the potential systemic side effects. For successful local delivery, the drug must be retained at the target site to control its release. To accomplish this goal, our study aimed to encapsulate MTZ in microsponges (MSPs) to sustain its release, and then loading these MSPs in thermosensitive in situ hydrogels to be retained in the periodontal pocket. Using this smart thermosensitive in situ gel, MTZ microsponges can achieve the advantages of drug reservoir and perform controlled drug release. Microsponges (MSPs) are polymeric delivery systems made up of microspheres, which are spherical porous particles (5 to 300 µm) and are capable of trapping a wide range of drugs, reducing side effects, and improving drug stability (Jyothi, Kumar et al. 2019 )(Jyoti and Kumar 2018 ). Microsponges are used mostly for topical use such as periodontitis treatment. In situ gels are liquid preparations that can be injected into the periodontal pocket and then solidifies to form a gel (Yadav, Kanwar et al. 2020 ). Because the infections are confined to the periodontal pockets or the oral cavity, localized intra pocket medicine delivery will be more effective than systemic treatment. 2. Materials and methods 2.1. Materials Metronidazole (MTZ) was obtained from Amriya Pharm Industries, (Alexandria, Egypt). Eudragit RS 100 (ERS 100), pluronic F-127 (PF-127), pluronic F-68 and hydroxy propyl methyl cellulose K100 (HPMC-K100) were purchased from Sigma Chemical Co. (St. Louis, USA). Mueller Hinton agar was obtained from Difco laboratories, Michigan, USA. Other chemicals were all of pharmaceutical grade and were used as received. 2.2. Preparation of Eudragit RS 100 microsponges (ERS-MSPs) The oil in oil emulsion solvent diffusion technique was used to prepare MTZ-MSPs (Jyoti and Kumar 2018 ) (Othman, Zayed et al. 2020 ). Briefly, after dissolving ERS 100 in acetone to obtain a transparent solution, MTZ solution was added, along with magnesium stearate. Then, the entire mixture was placed in an ultrasonic bath (at a 90-kHz frequency, 5 min) to achieve a homogeneous dispersion as shown in Table 1 . This mixture was poured into 150 mL of liquid paraffin formerly cooled to 10°C ± 0.5°C under mechanical stirring for 45 min. The obtained oil in oil emulsion was further stirred for another 30 min at 35°C ± 5°C. During this stirring time, acetone was evaporated and the solidified MSPs were filtered, washed five times with n-hexane and then dried for 12 hrs at room temperature. The obtained MSPs were stored in a desiccator for further use. Table 1 Composition and preparation conditions of different metronidazole loaded microsponges Code ERS 100 (gm) Drug: polymer ratio (w/w) Acetone (mL) Polymer: solvent ratio (w/v) Stirring rate (rpm) F1 0.25 1:1 2.5 1:10 500 F2 0.50 1:2 5.0 1:10 500 F3 0.75 1:3 7.5 1:10 500 F4 1.00 1:4 10 1:10 500 F5 1.00 1:4 15 1:15 500 F6 1.00 1:4 20 1:20 500 F7 1.00 1:4 20 1:20 1000 2.3. Characterization of Eudragit RS 100 microsponges (ERS-MSPs) During the formulation of MSPs, several factors, including drug to polymer ratio (1:1, 1:2, 1:3, 1:4), stirring rate (500 and 1000 rpm) and polymer to solvent ratio (1:10, 1:15, 1:20), were studied and characterized to select the optimized ERS-MSPs (Table 1 ). Drug loading (DL%) and entrapment efficiency (EE%) MSPs equivalent to 10 mg of MTZ was crushed carefully in a glass mortar, transferred to phosphate buffer (pH = 6.8) and stirred for 2 hours using a magnetic stirrer. Then, the sample was filtered, and the drug concentration was determined spectrophotometrically at λ max = 324 nm against blank. DL% & EE% were calculated using the following equations (1&2) DL%= \(\:\frac{Amount\:of\:drug\:loaded\:in\:microsponges}{Weight\:of\:microsponges}\:\) × 100 (1) EE% = \(\:\frac{Actual\:amount\:of\:drug}{Theortical\:amount\:of\:drug\:}\) × 100 (2) Particle size and size distribution Particle size and polydispersity index (PDI) of each sample were determined using laser light scattering technique (Horiba LA-300, Horiba Instruments, INC, USA) (Vernekar, Gude et al. 2019 ). Optimized Eudragit RS 100 microsponges (ERS-MSPs) Based on the abovementioned variables, the optimized formulation was selected and subjected to further evaluation. Shape and surface morphology of optimized MSPs were investigated with scanning electron microscope (SEM, JSM5400LV, Tokyo, Japan). Moreover, X-ray diffraction (XRD) was done to investigate the changes in the physical condition of the drug during formulation (Phillips PW1710 Model, Holland) (Rani 2017 ). Furthermore, differential scanning calorimetric (DSC) analysis was done using DSC-50 (Shimadzu, Japan). Fourier transform infrared (FTIR) spectroscopy was done using KBr disk technique (Mantry, Momin et al. 2022 ). 2.4. Preparation of microsponges in situ gel In situ gel of optimized MSPs were prepared using pluronic F-127 (PF-127). Plain i n situ gel was prepared by the cold method via dispersing PF-127 (15–25% w/v) in cold water (4°C) under magnetic stirring for 1 hour (Rangabhatla, Tantishaiyakul et al. 2016 ) (Rangabhatla, Tantishaiyakul et al. 2017 ). Then, the preparations were stored in a refrigerator overnight till complete dissolution. Similarly, the medicated in situ gel was prepared using the selected concentration of PF-127 combined with 2% w/v of PF-68 and 0.5% w/v of HPMC-K100. MTZ-MSPs (1% w/v) was added to the gel solution with continuous stirring. 2.5. Characterization of microsponges in situ gel The different in situ gel formulations were evaluated using gelation temperature, gelling capacity,and rheological behavior. Then, the optimized in situ gel formulation was subjected for additional in vitro evaluation including drug release, mucoadhesive strength, anti-microbial activity, and stability studies. Gelation temperature and gelling capacity : The gelation temperature ( T gel ) was determined by placing the formulation (2 mL) in a sealed test tube. The test tube was dipped in a water bath (25°C) and its temperature was increased by 1°C increments for 5 minutes (Fathalla, Vangala et al. 2017 ). T gel is the temperature at which the formulation remained immobile during test tube inversion. In vitro gelling capacity was calculated by inserting a drop of the formulation in a vial containing 2 mL of phosphate buffer (pH 6.8) and equilibrated at 37°C, the gelling capacity was evaluated by visual examination of the gel formation and the required time for the gel to form and dissolve (Patil, Kadam et al. 2015 ). Rheological behavior : The rheological behavior of different in situ gel formulations (25 mL) was tested by measuring the viscosity at shear rates of 100 rpm (Brookfield Programmable Rheometer, Model RVDV-III U, Brookfield, Engineering laboratories, INC, Middleboro, MA, USA) (Fathalla, Vangala et al. 2017 ). The samples were equilibrated at 4°C or 37°C, before measurement (Spindle no 95). In vitro drug release : In vitro drug release study was done using dialysis membrane method (Yadav, Mishra et al. 2012 ) (Nair and Anoop 2014 )(Obiedallah, Abdel-Mageed et al. 2018 ). Dialysis membrane was filled with 1 mL of the sample and then immersed in a beaker containing 200 mL of phosphate buffer (pH 6.8). The beaker was kept in a shaking water bath (37°C ± 0.5°C, 50 rpm). 5 mL of each sample was withdrawn at predetermined time intervals and replaced with phosphate buffer. Drug released was measured spectrophotometrically at λ max = 324 nm against blank similarly treated. Mucoadhesive strength : Mucoadhesion of the sample was evaluated by measuring the force required to detach formulation from a mucin disc using modified mucoadhesive force measuring device (Rangabhatla, Tantishaiyakul et al. 2017 )(ALI IBRAHIM, Ismail et al. 2012 ). Mucin discs were prepared and hydrated for 30 seconds using 5% mucin solution (Jones, Woolfson et al. 2000 ). The force necessary to detach the mucin disc from the sample, which was equilibrated at 37°C to be gel, was calculated (Rangabhatla, Tantishaiyakul et al. 2017 , Wróblewska, Szymańska et al. 2020 ) (ALI IBRAHIM, Ismail et al. 2012 ). Antimicrobial activity : The antimicrobial activity of the in situ gel was measured by the plate diffusion method on Muller-Hinton agar. Gram-positive bacteria such as Staphylococcus aureus and Gram-negative bacteria such as Escherichia coli (E. coli) were used in the study. The experiment was done in triplicate and the average value of the inhibition zone was recorded (Edsman, Carlfors et al. 1998 ). 2.6. In vivo evaluation Ethics statement The in vivo study was approved by the institution's animal ethical committee of Assiut University (approval number 04-2023-200632), Egypt. Periodontitis in mice was induced by the silk ligature method (Dharmawati 2019 ). After insertion of the ligature, wistar mice (200 ± 10gm) were kept for 14 days. Then, mice were divided into 4 groups (n = 20): group I (GP I) is the healthy group (-ve control group), group II (GP II) is the untreated periodontitis group (+ ve control); group III (GP III) is the group treated with the selected in situ gel for 7 days via intra-pocket syringe and group IV (GP IV) is the group treated with the marketed MTZ solution for 7 days via epigastric tube. After treatment, mice were anesthetized to collect blood samples, then mercifully sacrificed using carbon dioxide asphyxiation to collect oral tissues. The in vivo study was approved by the institution's animal ethical committee (approval number 04-2023-200632). Using blood samples, total and differential white blood cells (WBCs) counts were performed to evaluate the initial effects of MTZ on peripheral blood leukocytosis (Pejčić, Kesić et al. 2011 ). Histopathological studies were done via fixation of dental-alveolar segments in 10% buffered formalin and decalcified in 7% nitric acid for 3 days, followed by three weeks decalcification using 8% formic acid and 8% chlorohydric acid mixture (1:1) (Botelho, Martins et al. 2010 ). Then, tissues were cut, dehydrated, clarified, and embedded in paraffin wax. These blocks were sliced and stained with hematoxylin and eosin (H & E) and Masson`s trichome stain. 2.7. Statistical analysis Statistical analysis of data was performed using one-way ANOVA (GraphPad Prism software version 5). The P-value < 0.05 were considered statistically significant. 3. Results and discussion 3.1. Optimization of Eudragit RS 100 microsponges Different MSPs were prepared using oil in oil emulsion solvent diffusion method (Srivastava and Pathak 2012 ). The emulsion was prepared using volatile organic solvent as an internal phase that was allowed to evaporate slowly at a controlled rate with continuous stirring (Othman, Zayed et al. 2017 ). The effect of drug to polymer ratio was investigated in formulations F1-F4 (1:1, 1:2, 1:3 and 1:4, respectively). Additionally, the effect of polymer to solvent ratio was investigated in formulations F4-F6 (1:10, 1:15 and 1:20, respectively). Finally, the effect of stirring rate was studied in formulations F6 and F7 (500 and 1000 rpm, respectively). The effect of these different variables on EE%, DL% and particle size were summarized in Table 2 . Table 2 Effect of drug to polymer ratio, polymer to solvent ratio and stirring rate on the different MSPs formulations. Formulation code Entrapment efficiency % ± SD Drug loading % ± SD Particle size (µm) ± SD PDI ± SD F1 85.72 ± 1.982 32.97 ± 0.70 44.68 ± 1.03 0.315 ± 0.015 F2 87.84 ± 1.035 24.40 ± 1.87 57.36 ± 0.63 0.378 ± 0.024 F3 86.99 ± 3.462 18.91 ± 2.30 66.57 ± 0.27 0.376 ± 0.014 F4 88.76 ± 1.684 17.07 ± 1.08 66.31 ± 0.41 0.243 ± 0.039 F5 86.72 ± 0.577 14.95 ± 1.95 77.65 ± 2.65 0.246 ± 0.015 F6 87.02 ± 1.048 11.76 ± 1.34 77.95 ± 1.41 0.342 ± 0.035 F7 85.34 ± 1.750 11.53 ± 0.57 44.89 ± 0.81 0.162 ± 0.053 The results revealed that the different formulations resulted in EE > 80% (Table 2 ) and this could be attributed to full removal of water from the external phase with minimum drug loss and may be also attributed to the presence of nonpolar liquid paraffin in the external phase that inhibits the escape of the water-soluble drugs like MTZ (Page and Eke 2007 ). However, DL deceased significantly with decreasing the drug to polymer ratio, slightly with decreasing the polymer to solvent ratio and not affected with different stirring rates (Table 2 ). Regarding the effect of these variables on the particle size, it was noticed that size of MSPs significantly increased with increasing the polymer content and there was non-significant effect of polymer to solvent ratio on particle size. However, increasing the stirring rate significantly decreased the size of MSPs (Table 2 ). All MSPs had a good polydispersity index (PDI < 0.4). Magnesium stearate was tested as a stabilizer to prevent adherence between the particles of MSPs. The most appropriate concentration of magnesium stearate was 3% w/v. It has been reported that magnesium stearate reduced the interfacial tension and prevented the electrical charges and flocculation during MSPs preparation (Desavathu, Pathuri et al. 2017 ). Based on this optimization study, F7 was selected as the optimum MSPs with good EE (85.34 ± 1.75%), small particle size (44.89 ± 0.803 nm) and low PDI (0.162 ± 0.053). So, F7 was subjected to further evaluation using SEM, XRD, DSC and FTIR. Scanning electron microscopy (SEM) SEM showed spherical and uniform MSPs with several pores (Fig. 1 a). These pores were induced by solvent diffusion from the emulsion droplets during the formation of MSPs. The pores enable the dissolution medium to enter the MSPs to dissolve and release the entrapped drug from MSPs (Kumar and Ghosh 2015 ). Powder X-ray diffraction (PXRD) analysis X-ray diffraction pattern of MTZ showed peaks at 13.7°, 14.95°, 17.24°, 22.5°, 25.8° and 29.6° (Fig. 1 b) (Herculano, de Queiroz et al. 2011 ). MTZ-MSPs showed a hallow x-ray pattern similar to ERS 100 in plain formulation and this is may be due to entrapment of drug into polymer rather than amorphization (Mahaparale, Vinjamuri et al. 2019 ). Differential scanning calorimetry (DSC) DSC thermogram of MTZ showed endothermic peak at 163.83°C relative to the melting point of pure MTZ (Fig. 1 c) (Celebioglu and Uyar 2019 ). MSPs formulation didn’t change the nature of the drug, indicating that there is no incompatibility between the drug and the other additives (Fig. 1 c). Fourier transform infrared (FTIR) spectroscopy : Fig. 1 d shows a characteristic OH band at 3220 cm − 1 , C = CH; C-H stretch band at 3220.7 cm − 1 , peaks between 1300 to 1500 cm − 1 corresponding to NO₂ group, a characteristic peak at 1074 cm − 1 corresponding to C-OH; C-O stretch and a peak at 830 cm − 1 corresponding to C-NO₂; C-N stretch (Vecchi, Dos Santos et al. 2020 ). The FTIR spectrum of ERS 100 shows a characteristic C = O stretching band around 1712 cm − 1 (Srivastava, Kumar et al. 2012 ). FTIR spectrum of magnesium stearate shows a characteristic OH band form 2500 cm − 1 to 3300 cm − 1 , C = O stretch at 1700 cm − 1 , COO¯ asymmetric stretch at 1616 cm − 1 and CH₂ rocking at 720 cm − 1 . The FTIR results proved that MTZ is compatible with polymers and excipients of MSPs. 3.2. Optimization of microsponges in situ gels Optimized MSPs (F7) were added to different concentrations of PF-127 solutions. The different in situ gel formulations were evaluated via gelation temperature, gelling capacity, and viscosity. Gelation temperature ( T gel ) and gelling capacity : From our previous studies, it was found that 20–30% w/w of PF-127 had T gel below room temperature (Abd Ellah, Abouelmagd et al. 2018 ). However, PF-68 could increase the T gel to be free flowing at room temperature and gel at body temperature (Abd Ellah, Abouelmagd et al. 2018 ). Based on our studies, we formulated the selected MSPs with mixture of PF-127 and PF-68 in presence of the mucoadhesive polymer (HPMC). It was found that 20% PF-127 had T gel at 28.1°C ± 0.5°C, while 20% PF-127/2% PF-68 converted to gel at 35.4°C ± 0.5°C, which decreased to 33.8°C ± 0.5°C in presence of 0.5% HPMC (Table 3 ). This T gel (33.8°C ± 0.5°C) is acceptable to be free flowing at room temperature (25°C) and gel at buccal cavity (37°C) to treat periodontitis (Gurav and Husukale 2023 ). Gelling capacity of this formulation was also acceptable, it showed immediate gelation, which remained for hours (Patil, Kadam et al. 2015 ). Table 3 Gelation temperature and viscosity of the optimized in situ gels Formulation Code Gelation temperature (°C) Viscosity (cp) Shear rate=100rpm 4°C 37°C 20% PF-127 28.10 ± 0.5 1125 ± 2 14000 ± 3 20% PF-127/2% PF-68/0.5 HPMC 33.83 ± 0.5 1777 ± 2 23100 ± 1 Rheological behavior : Table 3 shows that the in situ gel exhibited low viscosity at low temperature (< 33°C) and with increasing temperature, the viscosity increased, and gel formed at oral physiological temperature (Gratieri, Gelfuso et al. 2010 ). Also, it was found that the viscosity was higher with addition of PF-68 and HPMC (Table 3 ). In vitro drug release Incorporation of free MTZ into pluronic-based in situ gel showed retardation of MTZ release (< 90% after 12 hours) (Fig. 2 A). Encapsulation of MTZ in MSPs (F7) significantly decreased its diffusion and release to the external phase (< 60% after 12 hrs) (Fig. 2 A). Additionally, incorporation of MTZ-MSPs into the pluronic-based in situ gelling formulation showed more retardation of drug release (< 45% after 12 hours). Based on this study, this in situ gel acts as an additional barrier to drug release. Presence of HPMC enhanced the viscosity and regulated the drug release capabilities (Chonkar, Nayak et al. 2015 ). Mucoadhesive strength Mucoadhesive strength is an important parameter for in situ forming gels to prevent rapid drainage and hence prolong residence time into the periodontal pocket. Mucoadhesive force of the plain gel was satisfactory (around 59 mN). This could be attributed to the hydrophilic oxide groups of pluronic polymer, which could bind to oligosaccharide chains (ALI IBRAHIM, Ismail et al. 2012 ). The in situ gel containing MTZ-MSPs showed significantly higher mucoadhesive strength (around 83 mN) (p < 0.0001) due to presence of positively charged Eudragit RS, which electrostatically interact with the mucin surface (Chaves, Frank et al. 2018 ). Antimicrobial activity MTZ-MSPs in situ gel showed a good anti-bacterial activity with a good inhibition zone. This may be attributed to the sustained release of MTZ from MSPs, which are loaded in the in situ gel. However, due to this sustained effect, it was found that the MTZ-MSPs in situ gel had significantly lower inhibition zone compared to MTZ solution (p < 0.05) (Fig. 2 B). Also, the results revealed that the plain in situ gel didn’t show any antimicrobial activity. 3.3. In vivo evaluation After 7 days of treatment with either MTZ solution or MTZ-MSPs in situ gel, blood samples and oral tissues were collected to determine the efficacy of the in situ gel. Total WBCs count is higher in patients with periodontitis compared to healthy patients (Al-Rasheed 2012 ). Similarly, it was found in this study that WBCs count was significantly higher in the untreated group (GP II, positive control group) compared to the other groups (Fig. 3 ) . However, marketed MTZ solution-treated group (GP IV) showed WBCs count like the untreated group (p = 0.2005). Accordingly, in situ gel had a remarkable efficacy in decreasing WBCs, indicating the decrease of periodontitis inflammation. This could be attributed to the efficient and localized intra-pocket administration of the thermosensitive in situ gel formulation, which was easily administered as liquid then rapidly converted into gel at oral cavity temperature (Abd Ellah, Abouelmagd et al. 2018 ). The gel has residence time higher than that of solutions (Abd Ellah, Abouelmagd et al. 2018 , Chaves, Frank et al. 2018 ). Histopathological studies were done on all the different groups. Control negative group (GP I) showed normal periodontal tissues with no signs of inflammation (Fig. 4 A & 5 A). In the untreated periodontitis group ( GP II , positive control), Figs. 4 B & C show severe inflammatory response to ligature thread irritation, which included gingival tissue, periodontal ligament, and alveolar bone. This positive control group showing signs of inflammation; congestion of the periodontal blood vessels (stars, Fig. 4 B).) and presence of fibrous tissue and infiltrated with inflammatory cell mainly lymphocytes (Fig. 4 C” arrows“ and Fig. 5 B ) . Also, an increase in inflammatory cells, such as neutrophils and lymphocytes, was found, as well as an increase in the number of fibroblast cells and osteoclast cells. Successful induction of periodontitis in positive control group was confirmed by fragments of thin trabecular bone and large medullary spaces due to depletion of connectedness between the bone tissues (Figs. 4 B &C) . However, the use of in situ gel in GP III restored architecture of trabecular bone with well-connected bone matrix and small medullary spaces with normal periodontal tissues and no signs of inflammation (Fig. 4 D & 5 C ). In contrast, group IV that have periodontitis treated with the marketed MTZ solution still showing congestion of blood vessels (stars, Fig. 4 E) and lymphocytes infiltration (arrows, Fig. 4 F ) with increased inflammatory cells in the periodontal tissues (Fig. 5 D). Systemic administration of MTZ can lead to an unnecessary load to other sites. Furthermore, the drugs' therapeutic effect at infected locations after systemic delivery is only temporary. As a result, longer periods and repeated dosing are required to obtain the desired results. However, MTZ in situ gel was delivered by intra-pocket injection, leading to rapid healing of the periodontal inflammation and alveolar bone compared to the marketed solution, which requires a longer period of administration and a more frequent doses in order to reach the beneficial therapeutic effect and complete healing for the periodontal tissues (Barat, Srinatha et al. 2007 ). Conclusion Metronidazole was successfully prepared as MTZ-MSPs and further incorporated into an in situ gelling system. These MSPs were uniform and spherical in shape, with small particle size (45 ± 1 µm) and sponge porous surface. The optimization process demonstrated that the encapsulation efficiency of MTZ is dependent on the type and amount of polymer and solvent. Formulation F7 (1:4 drug: polymer, 3%w/v magnesium stearate and 20 mL acetone) was selected as the optimum formulation (EE% of 87 ± 1, particle size of 45 ± 1 µm, PDI 0.2 ± 0.1). Additionally, F7 resulted in sustained release, 57 ± 1 through 12 hours. FTIR studies, powder x-ray diffraction and differential scanning calorimetry confirmed that there were no interactions between MTZ and the other excipients used in MSPs. MTZ-MSPs showed intrinsic antimicrobial activity. The porous nature of polymeric MSPs allowed MTZ to freely travel in and out of the particles exhibiting antimicrobial activity in the areas surrounding the periodontal pockets. The MTZ-MSPs were successfully incorporated into in situ gelling system for intra-pocket delivery. The selected in situ gel formulation was a solution at room temperature and gelled at oral cavity temperature, indicating ease of injection into periodontal pockets, followed by gel formation. Finally, the in vivo study revealed that the optimized in situ gel exhibited decrease in gingival inflammation, which is confirmed by the histopathological study of the periodontium. Declarations Conflict of interest The authors declare no conflict of interest. Institutional Review Board Statement The study was approved by the institution's animal ethical committee of Assiut University (approval number 04-2023-200632),Egypt. Informed Consent Statement : Not applicable. Data sharing statement No/Not applicable (this manuscript does not report data generation or analysis). Funding: none. Author Contribution All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work. References Abd Ellah NH, Abouelmagd SA, Abbas AM, Shaaban OM, Hassanein KM. Dual-responsive lidocaine in situ gel reduces pain of intrauterine device insertion. Int J Pharm. 2018;538(1–2):279–86. Al-Rasheed A. Elevation of white blood cells and platelet counts in patients having chronic periodontitis. Saudi Dent J. 2012;24(1):17–21. ALI IBRAHIM E-S, Ismail S, Fetih G, Shaaban O, Hassanein K, Abdellah NH. Development and characterization of thermosensitive pluronic-based metronidazole in situ gelling formulations for vaginal application. Acta Pharm. 2012;62(1):59–70. Barat R, Srinatha A, Pandit J, Anupurba S, Mittal N. Chitosan inserts for periodontitis: Influence of drug loading, plasticizer and crosslinking on metronidazole release. Acta Pharm. 2007;57(4):469–77. Botelho MA, Martins JG, Ruela RS, Queiroz DB, Ruela WS. Nanotechnology in ligature-induced periodontitis: protective effect of a doxycycline gel with nanoparticules. J Appl Oral Sci. 2010;18:335–42. Celebioglu A, Uyar T. Metronidazole/hydroxypropyl-β-cyclodextrin inclusion complex nanofibrous webs as fast-dissolving oral drug delivery system. Int J Pharm. 2019;572:118828. Ceruelos AH, Romero-Quezada L, Ledezma JR, Contreras LL. Therapeutic uses of metronidazole and its side effects: an update. Eur Rev Med Pharmacol Sci. 2019;23(1):397–401. Chaves PDS, Frank LA, Frank AG, Pohlmann AR, Guterres SS, Beck RCR. Mucoadhesive properties of Eudragit® RS100, Eudragit® S100, and Poly (ε-caprolactone) nanocapsules: influence of the vehicle and the mucosal surface. AAPS PharmSciTech. 2018;19:1637–46. Chonkar A, Nayak U, Udupa N. Smart polymers in nasal drug delivery. Indian J Pharm Sci. 2015;77(4):367. Desavathu M, Pathuri R, Chunduru M. Design, development and characterization of valsartan microsponges by quasi emulsion technique and the impact of stirring rate on microsponge formation. J Appl Pharm Sci. 2017;7(1):193–8. Dharmawati I. Pocket measurement methods in wistar rats periodontitis induced by bacteria and the installation of silk ligature: An experimental studies. Int J Appl Pharm. 2019;11(4):172–4. Edsman K, Carlfors J, Petersson R. Rheological evaluation of poloxamer as an in situ gel for ophthalmic use. Eur J Pharm Sci. 1998;6(2):105–12. Fathalla ZM, Vangala A, Longman M, Khaled KA, Hussein AK, El-Garhy OH, Alany RG. Poloxamer-based thermoresponsive ketorolac tromethamine in situ gel preparations: Design, characterisation, toxicity and transcorneal permeation studies. Eur J Pharm Biopharm. 2017;114:119–34. Gratieri T, Gelfuso GM, Rocha EM, Sarmento VH, de Freitas O, Lopez RFV. A poloxamer/chitosan in situ forming gel with prolonged retention time for ocular delivery. Eur J Pharm Biopharm. 2010;75(2):186–93. Gurav NH, Husukale PS. Development and Evaluation of In Situ Gel Formation for Treatment of Mouth Ulcer. Turkish J Pharm Sci. 2023;20(3):185. Herculano RD, de Queiroz AAA, Kinoshita A, Oliveira ON Jr, Graeff CF. On the release of metronidazole from natural rubber latex membranes. Mater Sci Engineering: C. 2011;31(2):272–5. Jones DS, Woolfson AD, Brown AF, Coulter WA, McClelland C, Irwin CR. Design, characterisation and preliminary clinical evaluation of a novel mucoadhesive topical formulation containing tetracycline for the treatment of periodontal disease. J Controlled Release. 2000;67(2–3):357–68. Jyothi KN, Kumar PD, Arshad P, Karthik M, Panneerselvam T. Microsponges: A Promising Novel Drug Delivery System. J Drug Delivery Ther. 2019;9(5–s):188–94. Jyoti J, Kumar S. Innovative and novel strategy: Microsponges for topical drug delivery. J Drug Delivery Ther. 2018;8(5):28–34. Kumar PM, Ghosh A. Development and evaluation of metronidazole loaded microsponge based gel for superficial surgical wound infections. J Drug Deliv Sci Technol. 2015;30:15–29. Mahaparale PR, Vinjamuri BP, Chavan MS, Chougule MB, Haware RV. Computational predictability of microsponge properties using different multivariate models. AAPS PharmSciTech. 2019;20(5):1–10. Mantry S, Momin A, Meher AD, Dilip DA, Balu VH, Dama GY. (2022). EMERGING IMPLEMENTATION OF DRUG LOADED WITH MICROSPONGES TECHNOLOGY AND THEIR ANTIFUNGAL ACTIVITY. J Pharm Negat Results: 835–48. Nair SC, Anoop K. Design and in vitro evaluation of controlled release satranidazole subgingival films for periodontitis therapy. Int J Pharm Sci Rev Res. 2014;24(1):8–14. Obiedallah MM, Abdel-Mageed A, Elfaham TH. Ocular administration of acetazolamide microsponges in situ gel formulations. Saudi Pharm J. 2018;26(7):909–20. Othman MH, Zayed GM, Ali UF, Abdellatif AA. Colon-specific tablets containing 5-fluorouracil microsponges for colon cancer targeting. Drug Dev Ind Pharm. 2020;46(12):2081–8. Othman MH, Zayed GM, El-Sokkary GH, Ali UF, Abdellatif AA, Othman M. Preparation and evaluation of 5-fluorouracil loaded microsponges for treatment of colon cancer. J Cancer Sci Ther. 2017;9(01):307–13. Page RC, Eke PI. Case definitions for use in population-based surveillance of periodontitis. J Periodontol. 2007;78:1387–99. Patil S, Kadam A, Bandgar S, Patil S. Formulation and evaluation of an in situ gel for ocular drug delivery of anticonjunctival drug. Cellul. Chem Technol. 2015;49:35–40. Pejčić A, Kesić L, Pešić Z, Mirković D, Stojanović M. White blood cell count in different stages of chronic periodontitis. Acta Clin Croatica. 2011;50(2):159–67. Rangabhatla ASL, Tantishaiyakul V, Boonrat O, Hirun N, Ouiyangkul P. Novel in situ mucoadhesive gels based on Pluronic F127 and xyloglucan containing metronidazole for treatment of periodontal disease. Iran Polym J. 2017;26(11):851–9. Rangabhatla ASL, Tantishaiyakul V, Oungbho K, Boonrat O. Fabrication of pluronic and methylcellulose for etidronate delivery and their application for osteogenesis. Int J Pharm. 2016;499(1–2):110–8. Rani TN, EVALUATION OF ONDANSETRON MICROSPONGES FOR TOPICAL APPLICATION.. Int J Res Biology Pharm. 2017;3(5):01–16. FORMULATION DEVELOPMENT AND. Srivastava R, Kumar D, Pathak K. Colonic luminal surface retention of meloxicam microsponges delivered by erosion based colon-targeted matrix tablet. Int J Pharm. 2012;427(2):153–62. Srivastava R, Pathak K. Microsponges: a futuristic approach for oral drug delivery. Expert Opin Drug Deliv. 2012;9(7):863–78. Suvan J, Leira Y, Moreno Sancho FM, Graziani F, Derks J, Tomasi C. Subgingival instrumentation for treatment of periodontitis. A systematic review. J Clin Periodontol. 2020;47:155–75. Vecchi CF, Dos Santos RS, Bruschi ML. Technological development of mucoadhesive film containing poloxamer 407, polyvinyl alcohol and polyvinylpyrrolidone for buccal metronidazole delivery. Therapeutic delivery. 2020;11(7):431–46. Vernekar A, Gude R, Ghadi N, Parab S, Shirodker A. (2019). Formulation and Characterization of Controlled Release Flurbiprofen Microsponges Loaded in Gels. Ind. J. Pharm. Edu. Res 53: S50â. Weir CB. Metronidazole. StatPearls [Internet]. StatPearls Publishing; 2022. Le. Wróblewska M, Szymańska E, Szekalska M, Winnicka K. (2020). Different types of gel carriers as metronidazole delivery systems to the oral mucosa. Polymers 12(3): 680. Wu C-z, Yuan Y-h, Liu H-h, Li S-s, Zhang B-w, Chen W. Z.-j. An, S.-y. Chen, Y.-z. Wu and B. Han (2020). Epidemiologic relationship between periodontitis and type 2 diabetes mellitus. BMC Oral Health 20: 1–15. Yadav R, Kanwar IL, Haider T, Pandey V, Gour V, Soni V. In situ gel drug delivery system for periodontitis: an insight review. Future J Pharm Sci. 2020;6(1):1–13. Yadav SK, Mishra S, Mishra B. Eudragit-based nanosuspension of poorly water-soluble drug: formulation and in vitro–in vivo evaluation. AAPS PharmSciTech. 2012;13(4):1031–44. Additional Declarations No competing interests reported. Supplementary Files graphicalabstract.png Graphical abstract Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8679532","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":585567719,"identity":"968d759c-8777-40fa-a566-053ac60aa865","order_by":0,"name":"Eman S. Abd El-Reheim","email":"","orcid":"","institution":"Badr University in Assiut","correspondingAuthor":false,"prefix":"","firstName":"Eman","middleName":"S. Abd","lastName":"El-Reheim","suffix":""},{"id":585567721,"identity":"e614dc72-d84e-4997-a0bc-4a79b7ca0685","order_by":1,"name":"Khaled M. A. Hassanein","email":"","orcid":"","institution":"Assiut University","correspondingAuthor":false,"prefix":"","firstName":"Khaled","middleName":"M. A.","lastName":"Hassanein","suffix":""},{"id":585567724,"identity":"77c20b49-0108-4418-a3d5-4e3a63253c1d","order_by":2,"name":"Helal F. Hetta","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYBACAwkILQcmeUjRYszARqqWxAaitZhL9xh//FFxJ33D/QbGB2/bGOz5GwhosZxzxkya58yz3A3HGJgN57YxJM44QMhhN3LMmBnbDoO0sEnztjEkMBChxfjjz3+H0w2OMbD/BmqxlydCi4EEb8PhBKAWNmagFsYNhLWklUnzHDtsOPNYYrPknHMSiRsJa0ne/PFHzWF5vsOHD354U2ZjL0dICxJgbAASEsSrHwWjYBSMglGAGwAAXQJAtmwvQxgAAAAASUVORK5CYII=","orcid":"","institution":"University of Tabuk","correspondingAuthor":true,"prefix":"","firstName":"Helal","middleName":"F.","lastName":"Hetta","suffix":""},{"id":585567727,"identity":"80cbdf2f-7b59-456a-aa62-3c751ecef604","order_by":3,"name":"Niveen G. Elgendy","email":"","orcid":"","institution":"Assiut University","correspondingAuthor":false,"prefix":"","firstName":"Niveen","middleName":"G.","lastName":"Elgendy","suffix":""},{"id":585567729,"identity":"4f0fdcf3-a7fa-4c4f-9acb-50463b492883","order_by":4,"name":"Gamal Eldin A. Elgendy","email":"","orcid":"","institution":"Assiut University","correspondingAuthor":false,"prefix":"","firstName":"Gamal","middleName":"Eldin A.","lastName":"Elgendy","suffix":""},{"id":585567735,"identity":"3fe10a5f-52c6-4cda-8879-f540fb9656ab","order_by":5,"name":"Abd Elrazaq A. Mohamed","email":"","orcid":"","institution":"Assiut University","correspondingAuthor":false,"prefix":"","firstName":"Abd","middleName":"Elrazaq A.","lastName":"Mohamed","suffix":""},{"id":585567739,"identity":"394ffaf2-63fb-41af-9e78-bc44098a0936","order_by":6,"name":"Noura H. Abd Ellah","email":"","orcid":"","institution":"Assiut University","correspondingAuthor":false,"prefix":"","firstName":"Noura","middleName":"H. Abd","lastName":"Ellah","suffix":""}],"badges":[],"createdAt":"2026-01-23 13:10:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8679532/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8679532/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101940731,"identity":"f77fcecd-7f40-4817-bdb9-5b8a2a21408b","added_by":"auto","created_at":"2026-02-05 09:16:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":396320,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCharacterization\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ethe optimum \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eEudragit microsponges\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e (MSPs, F7):\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ea) \u003c/strong\u003e\u003c/em\u003eScanning electron micrograph of MSPs at different magnification power (500x and1000x)\u003cem\u003e, \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb)\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003ePowder X-ray diffraction of metronidazole (A), plain MSPs (B), medicated MSPs (C)\u003cem\u003e, \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec)\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eDSC thermogram of metronidazole (A), Eudragit RS 100 (B), Magnesium stearate (C), Physical mixture of drug and polymer (D) and metronidazole MSPs (E)\u003cem\u003e, \u003c/em\u003e\u003cstrong\u003ed) \u003c/strong\u003eFTIR spectrum of MSPs, Eudragit (ERS 100), Magnesium stearate (Mg.st), physical mixture (Ph.mix) and metronidazole (MTZ).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8679532/v1/6876d5489a285721fa1c4b62.png"},{"id":101940729,"identity":"c4800325-376b-4027-ae1c-259221245547","added_by":"auto","created_at":"2026-02-05 09:16:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":63680,"visible":true,"origin":"","legend":"\u003cp\u003eA) \u003cem\u003eIn vitro\u003c/em\u003e release profile of MTZ from microsponges \u003cem\u003ein situ\u003c/em\u003e gel: F7 is MTZ microsponges, P7 is MTZ microsponges loaded in \u003cem\u003ein situ\u003c/em\u003e gel\u003cem\u003e,\u003c/em\u003e B) Inhibition zone of MTZ solution and MTZ-MSPs loaded \u003cem\u003ein situ\u003c/em\u003egel.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8679532/v1/0d2c704e2724830104dcfc9b.png"},{"id":101943674,"identity":"47f6b672-67f8-4aa3-85ad-5ee73c67a1a9","added_by":"auto","created_at":"2026-02-05 09:42:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":20786,"visible":true,"origin":"","legend":"\u003cp\u003eTotal white blood cells (WBCs) count for all groups: GP I: healthy group, GP II: untreated group, GP III: \u003cem\u003ein situ\u003c/em\u003e gel-treated group and GP IV: marketed MTZ solution group\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8679532/v1/668066551fcf8655673b4329.png"},{"id":101940734,"identity":"500d860b-4688-4e30-81ac-b8cfae9e2f55","added_by":"auto","created_at":"2026-02-05 09:16:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1141386,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative micrograph of mice periodontal tissue with H\u0026amp;E stain: Group I (A), group II (B,C), group III (D), group IV (E,F)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8679532/v1/05d82973fd5f75873e796a32.png"},{"id":101940733,"identity":"f0f885b7-02a3-4f17-bae2-5804941b5566","added_by":"auto","created_at":"2026-02-05 09:16:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1213521,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative micrograph of mice periodontal tissue stained by Masson's trichrome stain showing alveolar bone (Al) and periodontal tissue (P) from group I (A), group II (B), group III (C) and group IV (D).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8679532/v1/46b2811f35d68fda9a42b32a.png"},{"id":102962143,"identity":"cf97386a-c83a-467d-9d0d-742c278bffc8","added_by":"auto","created_at":"2026-02-19 04:03:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4355332,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8679532/v1/67e0eacb-99d4-4ba4-97ad-f38765755fbd.pdf"},{"id":101943633,"identity":"37e8686a-b037-4a12-9b6d-c995eb36eba5","added_by":"auto","created_at":"2026-02-05 09:42:36","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":555561,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-8679532/v1/e19f5eaccd96142899e83380.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antimicrobial and anti-inflammatory potential of thermoresponsive in situ gel loaded with metronidazole microsponges for localized treatment of periodontitis ","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePeriodontitis is an inflammatory condition that spreads deeply into the tissues, resulting in the loss of supporting connective tissue and alveolar bone. Periodontitis causes the formation of soft tissue pockets or widened fissures between the gingiva and the tooth root (Page and Eke \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). It is estimated that over 19% of adults globally suffer from severe periodontal disorders, contributing to over 1\u0026nbsp;billion cases worldwide (Wu, Yuan et al. 2020).\u003c/p\u003e \u003cp\u003eThe initial therapy for periodontitis is scaling and root planning (SRP), mechanical debridement of surfaces, and guidance on oral hygiene. Apart from conservative measures, antimicrobial agents can be used locally or systemically to treat periodontitis.\u003c/p\u003e \u003cp\u003eIt is challenging to provide antimicrobial agents systemically, which results in insufficient drug concentration in the periodontal pocket. Furthermore, using large doses of these drugs can have several negative effects. However, using these drugs locally in the pockets of periodontal disease is an advanced approach to suppress or eradicate the pathogenic microorganisms and also to regulate the inflammatory response in the tissues (Suvan, Leira et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMetronidazole (MTZ) is a frequently used antimicrobial agent to inhibit anaerobic microorganisms in periodontal disease and also to treat several infections such as trichomoniasis, giardiasis, amebiasis, parasitic infections and gastrointestinal infections (Ceruelos, Romero-Quezada et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e)(Weir and Le 2022). Local periodontal MTZ delivery is very advantageous, both in terms of increasing the concentration directly in the infected site and minimizing the potential systemic side effects. For successful local delivery, the drug must be retained at the target site to control its release.\u003c/p\u003e \u003cp\u003eTo accomplish this goal, our study aimed to encapsulate MTZ in microsponges (MSPs) to sustain its release, and then loading these MSPs in thermosensitive \u003cem\u003ein situ\u003c/em\u003e hydrogels to be retained in the periodontal pocket. Using this smart thermosensitive \u003cem\u003ein situ\u003c/em\u003e gel, MTZ microsponges can achieve the advantages of drug reservoir and perform controlled drug release.\u003c/p\u003e \u003cp\u003eMicrosponges (MSPs) are polymeric delivery systems made up of microspheres, which are spherical porous particles (5 to 300 \u0026micro;m) and are capable of trapping a wide range of drugs, reducing side effects, and improving drug stability (Jyothi, Kumar et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e)(Jyoti and Kumar \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Microsponges are used mostly for topical use such as periodontitis treatment. \u003cem\u003eIn situ\u003c/em\u003e gels are liquid preparations that can be injected into the periodontal pocket and then solidifies to form a gel (Yadav, Kanwar et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Because the infections are confined to the periodontal pockets or the oral cavity, localized intra pocket medicine delivery will be more effective than systemic treatment.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eMetronidazole (MTZ) was obtained from Amriya Pharm Industries, (Alexandria, Egypt). Eudragit RS 100 (ERS 100), pluronic F-127 (PF-127), pluronic F-68 and hydroxy propyl methyl cellulose K100 (HPMC-K100) were purchased from Sigma Chemical Co. (St. Louis, USA). Mueller Hinton agar was obtained from Difco laboratories, Michigan, USA. Other chemicals were all of pharmaceutical grade and were used as received.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of Eudragit RS 100 microsponges (ERS-MSPs)\u003c/h2\u003e \u003cp\u003eThe oil in oil emulsion solvent diffusion technique was used to prepare MTZ-MSPs (Jyoti and Kumar \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) (Othman, Zayed et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Briefly, after dissolving ERS 100 in acetone to obtain a transparent solution, MTZ solution was added, along with magnesium stearate. Then, the entire mixture was placed in an ultrasonic bath (at a 90-kHz frequency, 5 min) to achieve a homogeneous dispersion as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. This mixture was poured into 150 mL of liquid paraffin formerly cooled to 10\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C under mechanical stirring for 45 min. The obtained oil in oil emulsion was further stirred for another 30 min at 35\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u0026deg;C. During this stirring time, acetone was evaporated and the solidified MSPs were filtered, washed five times with n-hexane and then dried for 12 hrs at room temperature. The obtained MSPs were stored in a desiccator for further use.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComposition and preparation conditions of different metronidazole loaded microsponges\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCode\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eERS 100\u003c/p\u003e \u003cp\u003e(gm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDrug: polymer\u003c/p\u003e \u003cp\u003eratio (w/w)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAcetone\u003c/p\u003e \u003cp\u003e(mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePolymer: solvent\u003c/p\u003e \u003cp\u003eratio (w/v)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eStirring rate\u003c/p\u003e \u003cp\u003e(rpm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Characterization of Eudragit RS 100 microsponges (ERS-MSPs)\u003c/h2\u003e \u003cp\u003eDuring the formulation of MSPs, several factors, including drug to polymer ratio (1:1, 1:2, 1:3, 1:4), stirring rate (500 and 1000 rpm) and polymer to solvent ratio (1:10, 1:15, 1:20), were studied and characterized to select the optimized ERS-MSPs (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDrug loading (DL%) and entrapment efficiency (EE%)\u003c/strong\u003e \u003cp\u003eMSPs equivalent to 10 mg of MTZ was crushed carefully in a glass mortar, transferred to phosphate buffer (pH\u0026thinsp;=\u0026thinsp;6.8) and stirred for 2 hours using a magnetic stirrer. Then, the sample was filtered, and the drug concentration was determined spectrophotometrically at λ max\u0026thinsp;=\u0026thinsp;324 nm against blank. DL% \u0026amp; EE% were calculated using the following equations (1\u0026amp;2)\u003c/p\u003e \u003c/p\u003e \u003cp\u003eDL%= \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{Amount\\:of\\:drug\\:loaded\\:in\\:microsponges}{Weight\\:of\\:microsponges}\\:\\)\u003c/span\u003e\u003c/span\u003e\u0026times; 100 (1)\u003c/p\u003e \u003cp\u003eEE% = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{Actual\\:amount\\:of\\:drug}{Theortical\\:amount\\:of\\:drug\\:}\\)\u003c/span\u003e\u003c/span\u003e\u0026times; 100 (2)\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eParticle size and size distribution\u003c/strong\u003e \u003cp\u003eParticle size and polydispersity index (PDI) of each sample were determined using laser light scattering technique (Horiba LA-300, Horiba Instruments, INC, USA) (Vernekar, Gude et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eOptimized Eudragit RS 100 microsponges (ERS-MSPs)\u003c/strong\u003e \u003cp\u003eBased on the abovementioned variables, the optimized formulation was selected and subjected to further evaluation. Shape and surface morphology of optimized MSPs were investigated with scanning electron microscope (SEM, JSM5400LV, Tokyo, Japan). Moreover, X-ray diffraction (XRD) was done to investigate the changes in the physical condition of the drug during formulation (Phillips PW1710 Model, Holland) (Rani \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Furthermore, differential scanning calorimetric (DSC) analysis was done using DSC-50 (Shimadzu, Japan). Fourier transform infrared (FTIR) spectroscopy was done using KBr disk technique (Mantry, Momin et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Preparation of microsponges \u003cem\u003ein situ\u003c/em\u003e gel\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn situ\u003c/em\u003e gel of optimized MSPs were prepared using pluronic F-127 (PF-127). Plain i\u003cem\u003en situ\u003c/em\u003e gel was prepared by the cold method via dispersing PF-127 (15\u0026ndash;25% w/v) in cold water (4\u0026deg;C) under magnetic stirring for 1 hour (Rangabhatla, Tantishaiyakul et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) (Rangabhatla, Tantishaiyakul et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Then, the preparations were stored in a refrigerator overnight till complete dissolution. Similarly, the medicated \u003cem\u003ein situ\u003c/em\u003e gel was prepared using the selected concentration of PF-127 combined with 2% w/v of PF-68 and 0.5% w/v of HPMC-K100. MTZ-MSPs (1% w/v) was added to the gel solution with continuous stirring.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Characterization of microsponges \u003cem\u003ein situ\u003c/em\u003e gel\u003c/h2\u003e \u003cp\u003eThe different \u003cem\u003ein situ\u003c/em\u003e gel formulations were evaluated using gelation temperature, gelling capacity,and rheological behavior. Then, the optimized \u003cem\u003ein situ\u003c/em\u003e gel formulation was subjected for additional \u003cem\u003ein vitro\u003c/em\u003e evaluation including drug release, mucoadhesive strength, anti-microbial activity, and stability studies.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eGelation temperature and gelling capacity\u003c/b\u003e: The gelation temperature (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003egel\u003c/em\u003e\u003c/sub\u003e) was determined by placing the formulation (2 mL) in a sealed test tube. The test tube was dipped in a water bath (25\u0026deg;C) and its temperature was increased by 1\u0026deg;C increments for 5 minutes (Fathalla, Vangala et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003egel\u003c/em\u003e\u003c/sub\u003e is the temperature at which the formulation remained immobile during test tube inversion. \u003cem\u003eIn vitro\u003c/em\u003e gelling capacity was calculated by inserting a drop of the formulation in a vial containing 2 mL of phosphate buffer (pH 6.8) and equilibrated at 37\u0026deg;C, the gelling capacity was evaluated by visual examination of the gel formation and the required time for the gel to form and dissolve (Patil, Kadam et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eRheological behavior\u003c/b\u003e: The rheological behavior of different \u003cem\u003ein situ\u003c/em\u003e gel formulations (25 mL) was tested by measuring the viscosity at shear rates of 100 rpm (Brookfield Programmable Rheometer, Model RVDV-III U, Brookfield, Engineering laboratories, INC, Middleboro, MA, USA) (Fathalla, Vangala et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The samples were equilibrated at 4\u0026deg;C or 37\u0026deg;C, before measurement (Spindle no 95).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003edrug release\u003c/b\u003e: \u003cem\u003eIn vitro\u003c/em\u003e drug release study was done using dialysis membrane method (Yadav, Mishra et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) (Nair and Anoop \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e)(Obiedallah, Abdel-Mageed et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Dialysis membrane was filled with 1 mL of the sample and then immersed in a beaker containing 200 mL of phosphate buffer (pH 6.8). The beaker was kept in a shaking water bath (37\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C, 50 rpm). 5 mL of each sample was withdrawn at predetermined time intervals and replaced with phosphate buffer. Drug released was measured spectrophotometrically at λ\u003csub\u003emax\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;324 nm against blank similarly treated.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eMucoadhesive strength\u003c/b\u003e: Mucoadhesion of the sample was evaluated by measuring the force required to detach formulation from a mucin disc using modified mucoadhesive force measuring device (Rangabhatla, Tantishaiyakul et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)(ALI IBRAHIM, Ismail et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Mucin discs were prepared and hydrated for 30 seconds using 5% mucin solution (Jones, Woolfson et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The force necessary to detach the mucin disc from the sample, which was equilibrated at 37\u0026deg;C to be gel, was calculated (Rangabhatla, Tantishaiyakul et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Wr\u0026oacute;blewska, Szymańska et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) (ALI IBRAHIM, Ismail et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eAntimicrobial activity\u003c/b\u003e: The antimicrobial activity of the \u003cem\u003ein situ\u003c/em\u003e gel was measured by the plate diffusion method on Muller-Hinton agar. Gram-positive bacteria such as \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and Gram-negative bacteria such as \u003cem\u003eEscherichia coli (E. coli)\u003c/em\u003e were used in the study. The experiment was done in triplicate and the average value of the inhibition zone was recorded (Edsman, Carlfors et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1998\u003c/span\u003e).\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. \u003cem\u003eIn vivo\u003c/em\u003e evaluation\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eEthics statement\u003c/strong\u003e \u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e study was approved by the institution's animal ethical committee of Assiut University (approval number 04-2023-200632), Egypt.\u003c/p\u003e \u003c/p\u003e \u003cp\u003ePeriodontitis in mice was induced by the silk ligature method (Dharmawati \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). After insertion of the ligature, wistar mice (200\u0026thinsp;\u0026plusmn;\u0026thinsp;10gm) were kept for 14 days. Then, mice were divided into 4 groups (n\u0026thinsp;=\u0026thinsp;20): group I (GP I) is the healthy group (-ve control group), group II (GP II) is the untreated periodontitis group (+\u0026thinsp;ve control); group III (GP III) is the group treated with the selected \u003cem\u003ein situ\u003c/em\u003e gel for 7 days via intra-pocket syringe and group IV (GP IV) is the group treated with the marketed MTZ solution for 7 days via epigastric tube. After treatment, mice were anesthetized to collect blood samples, then mercifully sacrificed using carbon dioxide asphyxiation to collect oral tissues. The \u003cem\u003ein vivo\u003c/em\u003e study was approved by the institution's animal ethical committee (approval number 04-2023-200632). Using blood samples, total and differential white blood cells (WBCs) counts were performed to evaluate the initial effects of MTZ on peripheral blood leukocytosis (Pejčić, Kesić et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHistopathological studies were done via fixation of dental-alveolar segments in 10% buffered formalin and decalcified in 7% nitric acid for 3 days, followed by three weeks decalcification using 8% formic acid and 8% chlorohydric acid mixture (1:1) (Botelho, Martins et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Then, tissues were cut, dehydrated, clarified, and embedded in paraffin wax. These blocks were sliced and stained with hematoxylin and eosin (H \u0026amp; E) and Masson`s trichome stain.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis of data was performed using one-way ANOVA (GraphPad Prism software version 5). The P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Optimization of Eudragit RS 100 microsponges\u003c/h2\u003e \u003cp\u003eDifferent MSPs were prepared using oil in oil emulsion solvent diffusion method (Srivastava and Pathak \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The emulsion was prepared using volatile organic solvent as an internal phase that was allowed to evaporate slowly at a controlled rate with continuous stirring (Othman, Zayed et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The effect of drug to polymer ratio was investigated in formulations F1-F4 (1:1, 1:2, 1:3 and 1:4, respectively). Additionally, the effect of polymer to solvent ratio was investigated in formulations F4-F6 (1:10, 1:15 and 1:20, respectively). Finally, the effect of stirring rate was studied in formulations F6 and F7 (500 and 1000 rpm, respectively). The effect of these different variables on EE%, DL% and particle size were summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of drug to polymer ratio, polymer to solvent ratio and stirring rate on the different MSPs formulations.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFormulation\u003c/p\u003e \u003cp\u003ecode\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEntrapment\u003c/p\u003e \u003cp\u003eefficiency\u003c/p\u003e \u003cp\u003e% \u0026plusmn; SD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDrug\u003c/p\u003e \u003cp\u003eloading\u003c/p\u003e \u003cp\u003e% \u0026plusmn; SD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eParticle size\u003c/p\u003e \u003cp\u003e(\u0026micro;m)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePDI\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e85.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.982\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e32.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e44.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.315\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e87.84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.035\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e24.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e57.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.378\u0026thinsp;\u0026plusmn;\u0026thinsp;0.024\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e86.99\u0026thinsp;\u0026plusmn;\u0026thinsp;3.462\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e18.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e66.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.376\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e88.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.684\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e17.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e66.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.243\u0026thinsp;\u0026plusmn;\u0026thinsp;0.039\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e86.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.577\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e14.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e77.65\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.246\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e87.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e11.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e77.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.342\u0026thinsp;\u0026plusmn;\u0026thinsp;0.035\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e85.34\u0026thinsp;\u0026plusmn;\u0026thinsp;1.750\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e11.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e44.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.162\u0026thinsp;\u0026plusmn;\u0026thinsp;0.053\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe results revealed that the different formulations resulted in EE\u0026thinsp;\u0026gt;\u0026thinsp;80% (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and this could be attributed to full removal of water from the external phase with minimum drug loss and may be also attributed to the presence of nonpolar liquid paraffin in the external phase that inhibits the escape of the water-soluble drugs like MTZ (Page and Eke \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). However, DL deceased significantly with decreasing the drug to polymer ratio, slightly with decreasing the polymer to solvent ratio and not affected with different stirring rates (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Regarding the effect of these variables on the particle size, it was noticed that size of MSPs significantly increased with increasing the polymer content and there was non-significant effect of polymer to solvent ratio on particle size. However, increasing the stirring rate significantly decreased the size of MSPs (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). All MSPs had a good polydispersity index (PDI\u0026thinsp;\u0026lt;\u0026thinsp;0.4). Magnesium stearate was tested as a stabilizer to prevent adherence between the particles of MSPs. The most appropriate concentration of magnesium stearate was 3% w/v. It has been reported that magnesium stearate reduced the interfacial tension and prevented the electrical charges and flocculation during MSPs preparation (Desavathu, Pathuri et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBased on this optimization study, F7 was selected as the optimum MSPs with good EE (85.34\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75%), small particle size (44.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.803 nm) and low PDI (0.162\u0026thinsp;\u0026plusmn;\u0026thinsp;0.053). So, F7 was subjected to further evaluation using SEM, XRD, DSC and FTIR.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eScanning electron microscopy (SEM)\u003c/strong\u003e \u003cp\u003eSEM showed spherical and uniform MSPs with several pores (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). These pores were induced by solvent diffusion from the emulsion droplets during the formation of MSPs. The pores enable the dissolution medium to enter the MSPs to dissolve and release the entrapped drug from MSPs (Kumar and Ghosh \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePowder X-ray diffraction (PXRD) analysis\u003c/strong\u003e \u003cp\u003eX-ray diffraction pattern of MTZ showed peaks at 13.7\u0026deg;, 14.95\u0026deg;, 17.24\u0026deg;, 22.5\u0026deg;, 25.8\u0026deg; and 29.6\u0026deg; (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) (Herculano, de Queiroz et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). MTZ-MSPs showed a hallow x-ray pattern similar to ERS 100 in plain formulation and this is may be due to entrapment of drug into polymer rather than amorphization (Mahaparale, Vinjamuri et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDifferential scanning calorimetry (DSC)\u003c/strong\u003e \u003cp\u003eDSC thermogram of MTZ showed endothermic peak at 163.83\u0026deg;C relative to the melting point of pure MTZ (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) (Celebioglu and Uyar \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). MSPs formulation didn\u0026rsquo;t change the nature of the drug, indicating that there is no incompatibility between the drug and the other additives (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFourier transform infrared (FTIR) spectroscopy\u003c/b\u003e: Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed shows a characteristic OH band at 3220 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, C\u0026thinsp;=\u0026thinsp;CH; C-H stretch band at 3220.7 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, peaks between 1300 to 1500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponding to NO₂ group, a characteristic peak at 1074 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponding to C-OH; C-O stretch and a peak at 830 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponding to C-NO₂; C-N stretch (Vecchi, Dos Santos et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The FTIR spectrum of ERS 100 shows a characteristic C\u0026thinsp;=\u0026thinsp;O stretching band around 1712 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Srivastava, Kumar et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). FTIR spectrum of magnesium stearate shows a characteristic OH band form 2500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 3300 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, C\u0026thinsp;=\u0026thinsp;O stretch at 1700 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, COO\u0026macr; asymmetric stretch at 1616 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and CH₂ rocking at 720 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The FTIR results proved that MTZ is compatible with polymers and excipients of MSPs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Optimization of microsponges \u003cem\u003ein situ\u003c/em\u003e gels\u003c/h2\u003e \u003cp\u003eOptimized MSPs (F7) were added to different concentrations of PF-127 solutions. The different \u003cem\u003ein situ\u003c/em\u003e gel formulations were evaluated via gelation temperature, gelling capacity, and viscosity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGelation temperature\u003c/b\u003e (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003egel\u003c/em\u003e\u003c/sub\u003e) \u003cb\u003eand gelling capacity\u003c/b\u003e: From our previous studies, it was found that 20\u0026ndash;30% w/w of PF-127 had \u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003egel\u003c/em\u003e\u003c/sub\u003e below room temperature (Abd Ellah, Abouelmagd et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, PF-68 could increase the \u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003egel\u003c/em\u003e\u003c/sub\u003e to be free flowing at room temperature and gel at body temperature (Abd Ellah, Abouelmagd et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Based on our studies, we formulated the selected MSPs with mixture of PF-127 and PF-68 in presence of the mucoadhesive polymer (HPMC). It was found that 20% PF-127 had \u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003egel\u003c/em\u003e\u003c/sub\u003e at 28.1\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C, while 20% PF-127/2% PF-68 converted to gel at 35.4\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C, which decreased to 33.8\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C in presence of 0.5% HPMC (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This \u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003egel\u003c/em\u003e\u003c/sub\u003e (33.8\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C) is acceptable to be free flowing at room temperature (25\u0026deg;C) and gel at buccal cavity (37\u0026deg;C) to treat periodontitis (Gurav and Husukale \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Gelling capacity of this formulation was also acceptable, it showed immediate gelation, which remained for hours (Patil, Kadam et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGelation temperature and viscosity of the optimized \u003cem\u003ein situ\u003c/em\u003e gels\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFormulation\u003c/p\u003e \u003cp\u003eCode\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGelation\u003c/p\u003e \u003cp\u003etemperature (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eViscosity (cp)\u003c/p\u003e \u003cp\u003eShear rate=100rpm\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u0026deg;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37\u0026deg;C\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20% PF-127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e28.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1125\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e14000\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20% PF-127/2% PF-68/0.5 HPMC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e33.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1777\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e23100\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRheological behavior\u003c/b\u003e: Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows that the \u003cem\u003ein situ\u003c/em\u003e gel exhibited low viscosity at low temperature (\u0026lt;\u0026thinsp;33\u0026deg;C) and with increasing temperature, the viscosity increased, and gel formed at oral physiological temperature (Gratieri, Gelfuso et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Also, it was found that the viscosity was higher with addition of PF-68 and HPMC (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e drug release\u003c/strong\u003e \u003cp\u003eIncorporation of free MTZ into pluronic-based \u003cem\u003ein situ\u003c/em\u003e gel showed retardation of MTZ release (\u0026lt;\u0026thinsp;90% after 12 hours) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Encapsulation of MTZ in MSPs (F7) significantly decreased its diffusion and release to the external phase (\u0026lt;\u0026thinsp;60% after 12 hrs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Additionally, incorporation of MTZ-MSPs into the pluronic-based \u003cem\u003ein situ\u003c/em\u003e gelling formulation showed more retardation of drug release (\u0026lt;\u0026thinsp;45% after 12 hours). Based on this study, this \u003cem\u003ein situ\u003c/em\u003e gel acts as an additional barrier to drug release. Presence of HPMC enhanced the viscosity and regulated the drug release capabilities (Chonkar, Nayak et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMucoadhesive strength\u003c/strong\u003e \u003cp\u003eMucoadhesive strength is an important parameter for \u003cem\u003ein situ\u003c/em\u003e forming gels to prevent rapid drainage and hence prolong residence time into the periodontal pocket. Mucoadhesive force of the plain gel was satisfactory (around 59 mN). This could be attributed to the hydrophilic oxide groups of pluronic polymer, which could bind to oligosaccharide chains (ALI IBRAHIM, Ismail et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The \u003cem\u003ein situ\u003c/em\u003e gel containing MTZ-MSPs showed significantly higher mucoadhesive strength (around 83 mN) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) due to presence of positively charged Eudragit RS, which electrostatically interact with the mucin surface (Chaves, Frank et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAntimicrobial activity\u003c/strong\u003e \u003cp\u003eMTZ-MSPs \u003cem\u003ein situ\u003c/em\u003e gel showed a good anti-bacterial activity with a good inhibition zone. This may be attributed to the sustained release of MTZ from MSPs, which are loaded in the \u003cem\u003ein situ\u003c/em\u003e gel. However, due to this sustained effect, it was found that the MTZ-MSPs \u003cem\u003ein situ\u003c/em\u003e gel had significantly lower inhibition zone compared to MTZ solution (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Also, the results revealed that the plain \u003cem\u003ein situ\u003c/em\u003e gel didn\u0026rsquo;t show any antimicrobial activity.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. \u003cem\u003eIn vivo\u003c/em\u003e evaluation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAfter 7 days of treatment with either MTZ solution or MTZ-MSPs \u003cem\u003ein situ\u003c/em\u003e gel, blood samples and oral tissues were collected to determine the efficacy of the \u003cem\u003ein situ\u003c/em\u003e gel.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eTotal WBCs count is higher in patients with periodontitis compared to healthy patients (Al-Rasheed \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Similarly, it was found in this study that WBCs count was significantly higher in the untreated group (GP II, positive control group) compared to the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. However, marketed MTZ solution-treated group (GP IV) showed WBCs count like the untreated group (p\u0026thinsp;=\u0026thinsp;0.2005). Accordingly, \u003cem\u003ein situ\u003c/em\u003e gel had a remarkable efficacy in decreasing WBCs, indicating the decrease of periodontitis inflammation. This could be attributed to the efficient and localized intra-pocket administration of the thermosensitive \u003cem\u003ein situ\u003c/em\u003e gel formulation, which was easily administered as liquid then rapidly converted into gel at oral cavity temperature (Abd Ellah, Abouelmagd et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The gel has residence time higher than that of solutions (Abd Ellah, Abouelmagd et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Chaves, Frank et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHistopathological studies were done on all the different groups. Control negative group \u003cb\u003e(GP I)\u003c/b\u003e showed normal periodontal tissues with no signs of inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA \u0026amp; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In the untreated periodontitis group (\u003cb\u003eGP II\u003c/b\u003e, positive control), Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e\u0026amp; C\u003c/b\u003e show severe inflammatory response to ligature thread irritation, which included gingival tissue, periodontal ligament, and alveolar bone. This positive control group showing signs of inflammation; congestion of the periodontal blood vessels (stars, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).) and presence of fibrous tissue and infiltrated with inflammatory cell mainly lymphocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u0026rdquo; arrows\u0026ldquo; and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Also, an increase in inflammatory cells, such as neutrophils and lymphocytes, was found, as well as an increase in the number of fibroblast cells and osteoclast cells.\u003c/p\u003e \u003cp\u003eSuccessful induction of periodontitis in positive control group was confirmed by fragments of thin trabecular bone and large medullary spaces due to depletion of connectedness between the bone tissues (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB \u003cb\u003e\u0026amp;C)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eHowever, the use of \u003cem\u003ein situ\u003c/em\u003e gel in \u003cb\u003eGP III\u003c/b\u003e restored architecture of trabecular bone with well-connected bone matrix and small medullary spaces with normal periodontal tissues and no signs of inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD \u0026amp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn contrast, group \u003cb\u003eIV\u003c/b\u003e that have periodontitis treated with the marketed MTZ solution still showing congestion of blood vessels (stars, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) and lymphocytes infiltration (arrows, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e with increased inflammatory cells in the periodontal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eSystemic administration of MTZ can lead to an unnecessary load to other sites. Furthermore, the drugs' therapeutic effect at infected locations after systemic delivery is only temporary. As a result, longer periods and repeated dosing are required to obtain the desired results. However, MTZ \u003cem\u003ein situ\u003c/em\u003e gel was delivered by intra-pocket injection, leading to rapid healing of the periodontal inflammation and alveolar bone compared to the marketed solution, which requires a longer period of administration and a more frequent doses in order to reach the beneficial therapeutic effect and complete healing for the periodontal tissues (Barat, Srinatha et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eMetronidazole was successfully prepared as MTZ-MSPs and further incorporated into an \u003cem\u003ein situ\u003c/em\u003e gelling system. These MSPs were uniform and spherical in shape, with small particle size (45\u0026thinsp;\u0026plusmn;\u0026thinsp;1 \u0026micro;m) and sponge porous surface. The optimization process demonstrated that the encapsulation efficiency of MTZ is dependent on the type and amount of polymer and solvent. Formulation F7 (1:4 drug: polymer, 3%w/v magnesium stearate and 20 mL acetone) was selected as the optimum formulation (EE% of 87\u0026thinsp;\u0026plusmn;\u0026thinsp;1, particle size of 45\u0026thinsp;\u0026plusmn;\u0026thinsp;1 \u0026micro;m, PDI 0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1). Additionally, F7 resulted in sustained release, 57\u0026thinsp;\u0026plusmn;\u0026thinsp;1 through 12 hours. FTIR studies, powder x-ray diffraction and differential scanning calorimetry confirmed that there were no interactions between MTZ and the other excipients used in MSPs. MTZ-MSPs showed intrinsic antimicrobial activity. The porous nature of polymeric MSPs allowed MTZ to freely travel in and out of the particles exhibiting antimicrobial activity in the areas surrounding the periodontal pockets. The MTZ-MSPs were successfully incorporated into \u003cem\u003ein situ\u003c/em\u003e gelling system for intra-pocket delivery. The selected \u003cem\u003ein situ\u003c/em\u003e gel formulation was a solution at room temperature and gelled at oral cavity temperature, indicating ease of injection into periodontal pockets, followed by gel formation. Finally, the \u003cem\u003ein vivo\u003c/em\u003e study revealed that the optimized \u003cem\u003ein situ\u003c/em\u003e gel exhibited decrease in gingival inflammation, which is confirmed by the histopathological study of the periodontium.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eConflict of interest\u003c/strong\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eInstitutional Review Board Statement\u003c/h2\u003e \u003cp\u003eThe study was approved by the institution's animal ethical committee of Assiut University (approval number 04-2023-200632),Egypt.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eInformed Consent\u003c/strong\u003e \u003cp\u003e \u003cb\u003eStatement\u003c/b\u003e: Not applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eData sharing statement\u003c/strong\u003e \u003cp\u003eNo/Not applicable (this manuscript does not report data generation or analysis).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003enone.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbd Ellah NH, Abouelmagd SA, Abbas AM, Shaaban OM, Hassanein KM. Dual-responsive lidocaine in situ gel reduces pain of intrauterine device insertion. Int J Pharm. 2018;538(1\u0026ndash;2):279\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Rasheed A. Elevation of white blood cells and platelet counts in patients having chronic periodontitis. Saudi Dent J. 2012;24(1):17\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eALI IBRAHIM E-S, Ismail S, Fetih G, Shaaban O, Hassanein K, Abdellah NH. Development and characterization of thermosensitive pluronic-based metronidazole in situ gelling formulations for vaginal application. Acta Pharm. 2012;62(1):59\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarat R, Srinatha A, Pandit J, Anupurba S, Mittal N. Chitosan inserts for periodontitis: Influence of drug loading, plasticizer and crosslinking on metronidazole release. Acta Pharm. 2007;57(4):469\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBotelho MA, Martins JG, Ruela RS, Queiroz DB, Ruela WS. Nanotechnology in ligature-induced periodontitis: protective effect of a doxycycline gel with nanoparticules. J Appl Oral Sci. 2010;18:335\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCelebioglu A, Uyar T. Metronidazole/hydroxypropyl-β-cyclodextrin inclusion complex nanofibrous webs as fast-dissolving oral drug delivery system. Int J Pharm. 2019;572:118828.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCeruelos AH, Romero-Quezada L, Ledezma JR, Contreras LL. Therapeutic uses of metronidazole and its side effects: an update. Eur Rev Med Pharmacol Sci. 2019;23(1):397\u0026ndash;401.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChaves PDS, Frank LA, Frank AG, Pohlmann AR, Guterres SS, Beck RCR. Mucoadhesive properties of Eudragit\u0026reg; RS100, Eudragit\u0026reg; S100, and Poly (ε-caprolactone) nanocapsules: influence of the vehicle and the mucosal surface. AAPS PharmSciTech. 2018;19:1637\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChonkar A, Nayak U, Udupa N. Smart polymers in nasal drug delivery. Indian J Pharm Sci. 2015;77(4):367.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDesavathu M, Pathuri R, Chunduru M. Design, development and characterization of valsartan microsponges by quasi emulsion technique and the impact of stirring rate on microsponge formation. J Appl Pharm Sci. 2017;7(1):193\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDharmawati I. Pocket measurement methods in wistar rats periodontitis induced by bacteria and the installation of silk ligature: An experimental studies. Int J Appl Pharm. 2019;11(4):172\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdsman K, Carlfors J, Petersson R. Rheological evaluation of poloxamer as an in situ gel for ophthalmic use. Eur J Pharm Sci. 1998;6(2):105\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFathalla ZM, Vangala A, Longman M, Khaled KA, Hussein AK, El-Garhy OH, Alany RG. Poloxamer-based thermoresponsive ketorolac tromethamine in situ gel preparations: Design, characterisation, toxicity and transcorneal permeation studies. Eur J Pharm Biopharm. 2017;114:119\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGratieri T, Gelfuso GM, Rocha EM, Sarmento VH, de Freitas O, Lopez RFV. A poloxamer/chitosan in situ forming gel with prolonged retention time for ocular delivery. Eur J Pharm Biopharm. 2010;75(2):186\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGurav NH, Husukale PS. Development and Evaluation of In Situ Gel Formation for Treatment of Mouth Ulcer. Turkish J Pharm Sci. 2023;20(3):185.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerculano RD, de Queiroz AAA, Kinoshita A, Oliveira ON Jr, Graeff CF. On the release of metronidazole from natural rubber latex membranes. Mater Sci Engineering: C. 2011;31(2):272\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJones DS, Woolfson AD, Brown AF, Coulter WA, McClelland C, Irwin CR. Design, characterisation and preliminary clinical evaluation of a novel mucoadhesive topical formulation containing tetracycline for the treatment of periodontal disease. J Controlled Release. 2000;67(2\u0026ndash;3):357\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJyothi KN, Kumar PD, Arshad P, Karthik M, Panneerselvam T. Microsponges: A Promising Novel Drug Delivery System. J Drug Delivery Ther. 2019;9(5\u0026ndash;s):188\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJyoti J, Kumar S. Innovative and novel strategy: Microsponges for topical drug delivery. J Drug Delivery Ther. 2018;8(5):28\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar PM, Ghosh A. Development and evaluation of metronidazole loaded microsponge based gel for superficial surgical wound infections. J Drug Deliv Sci Technol. 2015;30:15\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMahaparale PR, Vinjamuri BP, Chavan MS, Chougule MB, Haware RV. Computational predictability of microsponge properties using different multivariate models. AAPS PharmSciTech. 2019;20(5):1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMantry S, Momin A, Meher AD, Dilip DA, Balu VH, Dama GY. (2022). EMERGING IMPLEMENTATION OF DRUG LOADED WITH MICROSPONGES TECHNOLOGY AND THEIR ANTIFUNGAL ACTIVITY. J Pharm Negat Results: 835\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNair SC, Anoop K. Design and in vitro evaluation of controlled release satranidazole subgingival films for periodontitis therapy. Int J Pharm Sci Rev Res. 2014;24(1):8\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eObiedallah MM, Abdel-Mageed A, Elfaham TH. Ocular administration of acetazolamide microsponges in situ gel formulations. Saudi Pharm J. 2018;26(7):909\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOthman MH, Zayed GM, Ali UF, Abdellatif AA. Colon-specific tablets containing 5-fluorouracil microsponges for colon cancer targeting. Drug Dev Ind Pharm. 2020;46(12):2081\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOthman MH, Zayed GM, El-Sokkary GH, Ali UF, Abdellatif AA, Othman M. Preparation and evaluation of 5-fluorouracil loaded microsponges for treatment of colon cancer. J Cancer Sci Ther. 2017;9(01):307\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePage RC, Eke PI. Case definitions for use in population-based surveillance of periodontitis. J Periodontol. 2007;78:1387\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatil S, Kadam A, Bandgar S, Patil S. Formulation and evaluation of an in situ gel for ocular drug delivery of anticonjunctival drug. Cellul. Chem Technol. 2015;49:35\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePejčić A, Kesić L, Pešić Z, Mirković D, Stojanović M. White blood cell count in different stages of chronic periodontitis. Acta Clin Croatica. 2011;50(2):159\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRangabhatla ASL, Tantishaiyakul V, Boonrat O, Hirun N, Ouiyangkul P. Novel in situ mucoadhesive gels based on Pluronic F127 and xyloglucan containing metronidazole for treatment of periodontal disease. Iran Polym J. 2017;26(11):851\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRangabhatla ASL, Tantishaiyakul V, Oungbho K, Boonrat O. Fabrication of pluronic and methylcellulose for etidronate delivery and their application for osteogenesis. Int J Pharm. 2016;499(1\u0026ndash;2):110\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRani TN, EVALUATION OF ONDANSETRON MICROSPONGES FOR TOPICAL APPLICATION.. Int J Res Biology Pharm. 2017;3(5):01\u0026ndash;16. FORMULATION DEVELOPMENT AND.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSrivastava R, Kumar D, Pathak K. Colonic luminal surface retention of meloxicam microsponges delivered by erosion based colon-targeted matrix tablet. Int J Pharm. 2012;427(2):153\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSrivastava R, Pathak K. Microsponges: a futuristic approach for oral drug delivery. Expert Opin Drug Deliv. 2012;9(7):863\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuvan J, Leira Y, Moreno Sancho FM, Graziani F, Derks J, Tomasi C. Subgingival instrumentation for treatment of periodontitis. A systematic review. J Clin Periodontol. 2020;47:155\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVecchi CF, Dos Santos RS, Bruschi ML. Technological development of mucoadhesive film containing poloxamer 407, polyvinyl alcohol and polyvinylpyrrolidone for buccal metronidazole delivery. Therapeutic delivery. 2020;11(7):431\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVernekar A, Gude R, Ghadi N, Parab S, Shirodker A. (2019). Formulation and Characterization of Controlled Release Flurbiprofen Microsponges Loaded in Gels. Ind. J. Pharm. Edu. Res 53: S50\u0026amp;#226.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeir CB. Metronidazole. StatPearls [Internet]. StatPearls Publishing; 2022. Le.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWr\u0026oacute;blewska M, Szymańska E, Szekalska M, Winnicka K. (2020). Different types of gel carriers as metronidazole delivery systems to the oral mucosa. Polymers 12(3): 680.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu C-z, Yuan Y-h, Liu H-h, Li S-s, Zhang B-w, Chen W. Z.-j. An, S.-y. Chen, Y.-z. Wu and B. Han (2020). Epidemiologic relationship between periodontitis and type 2 diabetes mellitus. BMC Oral Health 20: 1\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYadav R, Kanwar IL, Haider T, Pandey V, Gour V, Soni V. In situ gel drug delivery system for periodontitis: an insight review. Future J Pharm Sci. 2020;6(1):1\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYadav SK, Mishra S, Mishra B. Eudragit-based nanosuspension of poorly water-soluble drug: formulation and in vitro\u0026ndash;in vivo evaluation. AAPS PharmSciTech. 2012;13(4):1031\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Metronidazole, Microsponges, Periodontal disease, In situ gel","lastPublishedDoi":"10.21203/rs.3.rs-8679532/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8679532/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Periodontitis is an inflammatory disease that goes deeply into the tissues, causing loss of alveolar bone and supporting connective tissues. Severe periodontal diseases are estimated to affect around 19% of the global adult population, representing more than 1\u0026nbsp;billion cases worldwide. Metronidazole (MTZ) is frequently used to inhibit anaerobic bacteria in periodontal disease. This study aimed to fabricate MTZ-eudragit microsponges (MSPs) and then loading them into gel to evaluate its sustained local action in the buccal cavity. To optimize MTZ- MSPs, different variables such as drug:polymer ratio, polymer:solvent ratio and stirring rate were studied. characterization of MSPs, such as thermal behavior, surface morphology, particle size and drug release, were studied. The optimized MSPs were spherical with numerous pores on the surface (sponge-like particles). These optimized MSPs also showed high entrapment efficiency (87\u0026thinsp;\u0026plusmn;\u0026thinsp;1%), mean particle size of 45\u0026thinsp;\u0026plusmn;\u0026thinsp;1 \u0026micro;m (PDI 0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1) and sustained drug release (57\u0026thinsp;\u0026plusmn;\u0026thinsp;1% through 12 hours). The optimized MSPs were then incorporated into a thermoresponsive gel (20% PF-127/2% PF-68/0.5% HPMC). MSPs-containing gel was free flowing at room temperature and gel in the buccal cavity, retarding drug release and enhancing the mucoadhesion and antibacterial activity. studies on rats revealed that this gel is an effective and innovative approach to treating periodontitis. Conclusion, MSPs-loaded gel is a promising vehicle for MTZ to treat periodontitis.","manuscriptTitle":"Antimicrobial and anti-inflammatory potential of thermoresponsive in situ gel loaded with metronidazole microsponges for localized treatment of periodontitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-05 09:16:44","doi":"10.21203/rs.3.rs-8679532/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"40128daa-7b8d-47c8-9ac8-464eec5b7841","owner":[],"postedDate":"February 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-10T10:38:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-05 09:16:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8679532","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8679532","identity":"rs-8679532","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}