*4.3. Viscosity and Rheology Characterization*

The apparent viscosity values were measured using a viscometer with a cone-plate called the RM 100 CP2000 plus, which was manufactured by Lamy Rheology Instruments Company and is located in Champagne-au-Mont-d'Or, France. To carry out this measurement, a series of measurements at varying shear rates were taken at regular 15-s intervals during the equilibration time. This instrument was also used to test the rheological behavior of the substance. At room temperature, the shear stress of the formulations was determined by conducting the experiment at a variety of shear rates. The experiments were carried out three times to ensure accuracy.

### *4.4. Contact Angle*

The goniometer (FTA 1000, First Ten Angstroms, Newark, CA, USA) was used to determine the contact angle of the formula on various surfaces, such as glass, paraffin, and agarose gel, at a time point of 5 s after a pump out rate of 1.9 L/s. The manufacturer of the goniometer is First Ten Angstroms. After that, an estimate of the contact angle was derived from the very first automatic image of a droplet that was taken in triplicate (n = 3).


**Table 4.** Composition formula of 5–25% various Eudragit L, S in monopropylene glycol (A,B); and levofloxacin HCl-loaded 10–20% Eudragit L-based in monopropylene glycol.

### *4.5. Injectability*

Using the compression mode of a texture analyzer, a measurement of injectability was carried out in order to determine the precision of injection through the stainless needle (TA.XT plus, Stable Micro Systems, Godalming, UK). This apparatus was utilized to measure the pressure exerted during the injection of test liquid from a 1 mL syringe that was coupled to an 18-gauge needle. This device's upper probe pressed the syringe plunger with a constant force of 0.1 N and a speed of 1.0 mm/second until it reached the base of the syringe barrel. After analyzing force-displacement profiles, the maximum injection force, along with its associated energy for injection, was logged. Three separate trials of these experiments were carried out.

### *4.6. Mechanical Properties*

A texture analyzer was utilized in order to investigate the formulation's mechanical properties after it had been prepared (TA.XT Plus, Stable Micro Systems Ltd., Godalming, UK). After setting with a complete phase transformation into Eudragit matrix for 72 h, the prepared 150 microliters formulation was added into 0.6 percent *w*/*w* agarose gel. This process took place for 72 h. After that, an analytical probe from the previously described instrument was lowered into the polymer matrix at a rate of 0.5 mm per second. After maintaining this position for sixty seconds, the probe was then moved upward at a rate of ten millimeters per second. The amount of force that was checked at the point where the probe was able to penetrate the polymer matrix to its maximum depth was indicated as the maximum deformation force or the hardness of the material. Additionally, the amount of

force that was checked at the point where the probe was able to move upward between the surface of the sample and the probe was indicated as the adhesion force. The maximum deformation force, also known as F max deformation, is the force that is measured at the point where the probe has penetrated the specimen the farthest, whereas the remaining force, also known as F remaining, is a force that is measured after the specimen has been held for sixty seconds. A measure for the elasticity and plasticity of the specimen was the ratio "F remaining/F max deformation". Values of elasticity that are high indicate a material with high elasticity, while values of elasticity that are low indicate a material with high plasticity [61]. The experiments were carried out in triplicate.

### *4.7. Gel Formation Study*

The apparent gel formation at macroscopic level was performed by injected the prepared ISG through an 18-gauge needle into PBS (pH 6.8) medium. The cloudy gel or matrix-like formation at various times was observed (1, 3, 5, 15, and 30 min). Moreover, another gel formation behavior investigation at macroscopic level was also investigated. The in situ formation in an agarose well was also investigated under a stereoscope. This experiment was set to simulate the change of formulation within a periodontal pocket. To begin, an agarose gel with a height of one centimeter was prepared by dissolving 0.6 percent agarose in PBS with a pH of 6.8. The gel was then poured onto Petri dishes for the setting process. The settle agarose was drilled using a stainless cylinder cup (7.6 mm diameter) to create an agarose well, the cylindrical well with a capacity of 300 microliters that simulated a periodontal pocket. For the purpose of testing, a prepared formulation containing 150 microliters was inserted into the well which is a simulated periodontal pocket. After being exposed to PBS from the agarose gel, the phase separation of Eudragit leads to the generation of a thicker, cloudier gel or matrix-like substance over time. Under a stereo microscope (SZX10, Olympus Corp., Tokyo, Japan), photographs of the morphological Eudragit gel or matrix were taken simultaneously at 1, 3, 5, 15, and 30 min. The software used for these photographs was the SZX10 Series software (Olympus Corp., Tokyo, Japan).

### *4.8. Interfacial Phenomena of Formulation-Aqueous Phase*

Both a plain agarose gel with a weight-to-volume ratio of 0.6 percent and an agarose gel loaded with 0.4 µg/mL of SF were prepared. The LL10M, 0.4 µg/mL SF-loaded MP, 0.4 µg/mL SF-loaded LL10M, 3 µg/mL nile red (NR) -loaded MP, and 3 µg/mL nile red (NR) -loaded L10M were prepared. The different levels of color intensity after the emission of these fluorescent dyes were the root cause of the difference in concentration between them. Based on preliminary inspections, it was found that their concentration could be clearly seen at an intensity that was sufficient to observe the change in color or movement. In order to investigate the interfacial interaction between an aqueous phase and a solvent or ISG formulation, the SF-free and SF-loaded agarose gels were prepared, and then 50 microliters of prepared test samples were dropped close to that agarose gel. The interface interaction was investigated using an inverted fluorescent microscope (TE-2000U, Nikon, Kaw, Japan) by capturing the image using a blue (B2A) filter with excitation at 450–490 nm for probing the green color of SF and using a green (G2A) filter with excitation of 510–560 nm for tracking the red color of NR. Both of these filters were used in conjunction with the appropriate wavelengths of illumination.

### *4.9. Drug Content and In Vitro Drug Release Studies*

Using a UV-Visible spectrophotometer (Cary 60 UV-Vis, Model G6860A, Agilent, Selangor, Malaysia), a standard curve was constructed to estimate the amount of drug in the LVM, LLM10, LLM15, and LLM20 samples (n = 6). The in vitro drug release behavior of levofloxacin HCl from a developed formulation comprising 10, 15, and 20 percent Eudragit L was undertaken to employ the cup method to mimic the drug liberation behavior from the periodontal pocket. The drug release profiles that were obtained were compared with those obtained from the control formulation, which consisted of levofloxacin HCl dissolved

in monopropylene glycol at a concentration of one percent by weight. In a nutshell, 0.4 g of the refined dosage form was carefully placed into a cylindrical-shaped porcelain cup with 50 mL of PBS with a pH of 6.8 and heated to 37 degrees Celsius (diameter of 1 cm and higher of 1.2 cm). After that, it was placed in a rotation shaker (Model NB-205, N-Biotek, Gyeonggi-do, Republic of Korea) for 14 days at a temperature of 37 degrees Celsius and a rotational speed of 50 revolutions per minute. After that, 5 mL of release fluid was taken, and it was subsequently replaced with 5 mL of fresh PBS. The results of the study on the release of levofloxacin HCl were converted into a percentage of the cumulative drug amount using a UV-Visible spectrophotometer (Cary 60 UV-Vis, Model G6860A, Agilent, Selangor, Malaysia). The experiments were carried out three times to ensure accuracy.

Their drug release data were then fitted with a variety of mathematical models, such as zero order, first order, Higuchi's, and Korsmeyer–Peppas models, using the DD-Solver software application, which is an add-in program for Microsoft Excel. This enabled the researchers to determine the kinetics of drug release (Microsoft Corporation, Redmond, WA, USA). The estimated parameters, in particular the r<sup>2</sup> and model selection criteria (msc) values, were reported. Additionally, the release exponent (n-value) from the Korsmeyer– Peppas equation was used to indicate the mechanism of drug release. The following equations were used for mathematical model [93–95]. The zero-order model: the following equation indicates the release rate of the drug remains constant over time, independent of the drug concentration.

$$\mathbf{Q}\_{\mathbf{t}} = \mathbf{Q}\_{0} + \mathbf{k}\_{0}\mathbf{t} \tag{1}$$

where Q<sup>t</sup> is the total amount of drug release at given time (t), Q<sup>0</sup> is initial drug release, k<sup>0</sup> is the release rate constant, and t is the given time.

The first-order model is another commonly employed mathematical model for describing drug release behavior. It assumes that the release rate is proportional to the remaining drug concentration as following equation.

$$
\log \mathbf{Q}\_1 = \log \mathbf{Q}\_0 + \frac{\mathbf{k}\_1 \mathbf{t}}{2.303} \tag{2}
$$

where Q<sup>1</sup> is the amount of active agent released on time (t), Q<sup>0</sup> is the initial amount of drug dissolved, and k<sup>1</sup> is the first-order constant.

The Higuchi model assumes that drug release from polymer matrix systems is solely diffusion-controlled and that the drug is uniformly distributed in non-degradable planar systems. The Higuchi's model is given below.

$$\mathbf{Q}\_{\mathbf{t}} = \mathbf{k}\sqrt{\mathbf{t}} \tag{3}$$

where Q<sup>t</sup> is the total amount of drug release at given time (t), k is the release rate constant, and t is given time.

The Korsmeyer–Peppas model is widely used to assess the release mechanism. It incorporates a parameter 'n' to characterize the release mechanism, with values indicating different release kinetics. For swelling system, Fickian diffusion is indicated when the value of n is close to 0.45. If the value exceeds 0.45 but is lower than 0.89, this indicates non-Fickian diffusion or anomalous transport, and case II transport is indicated when n is close to 0.89. Fickian diffusion is a drug transport mechanism where structure relaxation is slower than diffusion. When relaxation exceeds diffusion, case-II transport occurs. Anomalous diffusion (non-Fickian transport) occurs when this drug release rate is close to structure relaxation [96–99].

$$\log \frac{\mathbf{M}\_{\text{(i-l)}}}{\mathbf{M}\_{\text{os}}} = \log \mathbf{K} + \mathbf{n} \log(\mathbf{t} - \mathbf{l}) \tag{4}$$

where M<sup>∞</sup> is the amount of drug at the equilibrium state, M<sup>i</sup> is the amount of drug released over time (t), K is the constant of incorporation of structural modifications and geometrical characteristics of the system (also considered the release velocity constant), n is the exponent of release (related to the drug release mechanism) in function of time t, and l is the latency time.

### *4.10. Scanning Electron Microscopy (SEM)*

After seven days of drug release testing in PBS with a pH of 6.8, the Eudragit-based ISG remnants were washed with 200 mL of distilled water and then freeze-dried with the assistance of a freeze dryer (TriadTM Labconco, Kansas City, MO, USA). The following process took place after the remnants had been stored in a desiccator for one week. The dried Eudragit-based ISG remnants were then coated with gold before being examined using the SEM method and admitted of comparison with intact levofloxacin HCl powder. At an accelerating voltage of 15 kV, a scanning electron microscope (SEM) TESCAN MIRA3 from Brno-Kohoutovice, Czech Republic, was used to observe the surface and cross-sectional morphologies of a dried Eudragit-based ISG remnant from a developed ISG system.

### *4.11. In Vitro Degradation Test*

The in vitro degradability of forming matrices was carried out by determining the mass loss of the system after the drug release test. The initial weight of the sample and that after the release test at 14 days were recorded and calculated (n = 3) as follows:

$$\% \text{ mass loss} = \left(\frac{\text{W}\_{\text{i}} - \text{W}\_{\text{l}}}{\text{W}\_{\text{i}}}\right) \times 100\tag{5}$$

where

Wi*=* initial weight of the sample W<sup>t</sup> = weight of remained sample at a specific time

### *4.12. X-ray Imaging and X-ray Tomographic Microscopy*

The remnants of LL15M and LL20M were collected from drug release study, then washed with distilled water. They were dried together using a freeze dryer. The dried systems were kept in a desiccator for 7 days. The dried systems were scrutinized with X-ray imaging and X-ray tomographic microscopy at the X-ray tomographic microscopy (XTM) beamline, Synchrotron Light Research Institute (SLRI), Thailand. The following conditions were set: Generation of X-ray beam was archived by 2.2-T multipole wiggler at the 1.2-GeV Siam Photon Source facility (150 mA). The synchrotron radiation with X-ray tomographic microscopy (SRXTM) inspections was performed using a filtered polychromatic X-ray beam at a mean energy of 11.5 kV with a source-to-sample distance of 34 m. Detection system was equipped with a 200 µm thick scintillator (YAG: Ce, Crytur, Turnov, Czech Republic), lens-coupled X-ray microscope, and the CMOs camera (PCO edge 5.5, 2560 pixels, 16 bits) (Optique Peter, Lentilly, France). Isotropic voxel size of 3.61 µm was used. X-ray projection was normalized using the Octopus reconstruction (TESCAN). The tomographic volumes were achieved via Drishti software (National Computational Infrastructure's VizLab). The porosity was determined using Octopus Analysis (TESCAN) [100].

### *4.13. Antimicrobial Activities*

The antimicrobial activities of monopropylene glycol, drug-free, and levofloxacin HCl-loaded Eudragit-based ISG formulations were evaluated against standard microbes (*S. aureus* DMST 6532, *MRSA S. aureus* ATCC 4430, *S. aureus* ATCC 6532, and *S. aureus* ATCC 25923), *E. coli* ATCC 8739, *C. albicans* 10231, *P. gingivalis* and *A. actinomycetemcomitans*) using the agar diffusion assay (cylinder plate method). For this bioactivity test, bacteria inocula were incubated for 36 h in tryptic soy broth (TSB), whereas sabouraud dextrose broth (SDB) was employed for *C. albicans*. The turbidity of broth suspensions of organisms was calibrated using the 0.5 McFarland standard. Consequently, the attained broth suspensions of *S. aureus* DMST 6532, *S. aureus* ATCC 4430, *S. aureus* ATCC 6532, *S. aureus* ATCC 25923, and *E. coli* ATCC 8739 were swab-spread on the tryptic soy agar plates, whilst sheep blood agar

and chocolate agar were used as media for anaerobic antimicrobial testing of *P. gingivalis* and *A. actinomycetemcomitans*, respectively. The inoculum of calibrated *C. albicans* was swab spread on sabouraud dextrose agar (SDA). The 200 microliters aliquot sample was filled into a sterilized cylindrical cup that was already placed on the surface of the swabbed agar before incubation at 37 ◦C for 24 h. In the case of antibacterial measurement against *P. gingivalis* and *A. actinomycetemcomitans,* the incubation was conducted in an anaerobic incubator (Forma Anaerobic System, Thermo Scientific, Ohio, USA) at 37 ◦C for 72 h. To indicate and compare the antimicrobial activities (n = 3), the diameter (mm) of the inhibition zone was measured individually with a ruler.

### *4.14. Fourier Transform Infrared (FTIR) Spectroscopy*

In addition to that, an FTIR spectrophotometer was used in order to record the spectra of the samples. The sample was mixed with potassium bromide, and then it was compressed with a plunger and die in a hydraulic press (pressure of 5 tons). The obtained pellet was placed inside of an FTIR chamber after it had been mounted. The sample's transmittance as a percentage was measured and recorded. In the range of 400–4000 cm−<sup>1</sup> , the spectra were collected at a resolution of 2 cm−<sup>1</sup> . .

### *4.15. Statistical Analysis*

SPSS for Windows (version 11.5) was employed for the statistical analysis. The difference between the experiments was determined by using Student's *t*-test. The results obtained were statistically significant because of the *p*-value, which was found to be less than 0.05.

**Author Contributions:** Experiments were conceived of and planned by S.S., S.T., T.C. and N.L., who were also responsible for sample preparation and evaluations. X-ray imaging and X-ray tomographic microscopy were both performed in the study that C.R. and S.S. carried out. S.S., N.L., S.T. and T.P. all made contributions to the interpretation of the results, with S.S. taking the lead in terms of writing the manuscript. Writing, reviewing, and editing were all tasks that T.C., S.T. and T.P. contributed to. T.P. and T.C. were a part of the planning process as well as the supervision of the work. Each author was responsible for providing constructive criticism and made significant contributions to the development of the research, the analysis, and the manuscript. The final, published version of the manuscript has been reviewed and accepted by all of the authors. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable. This research did not involve humans or animals for investigation.

**Informed Consent Statement:** Not applicable.

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

**Acknowledgments:** The authors would like to express their appreciation to the Synchrotron Light Research Institute, Center of Excellent in Pharmaceutical Nanotechnology, and Faculty of Pharmacy at Chiang Mai University for their generous support and facilitation of this study. In addition, we would like to express our gratitude to the Research and Creative Fund, which is part of the Silpakorn University Faculty of Pharmacy, for providing us with the opportunity to use their space and for their careful proofreading of this paper.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


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