(T1330-01-04) Characterization and In Vivo Evaluation of Oil Based Anti-adhesion Agent with Xanthan Gum

Purpose: The purpose of this study is to increase the anti-adhesive effect using xanthan gum. Xanthan gum has high viscosity, good water solubility, good thixotropic fluidity, and good biocompatibility. It can be used to slow down the absorption rate and lengthening the duration of the formulations in vivo. In this study, oil-in-water emulsion agents with oil droplets dispersed in different proportions of a xanthan gum hydrogel are prepared and evaluated.

Methods: Xanthan gum was chosen as a polymer that has biocompatibility and can adhere to abdominal cavity. The emulsion formulations to be named No.1, No.2, No.3, No.4 were prepared by varying the ratio of xanthan gum. Xanthan gum ratio of No.1 was 0.5% and it increased by 0.5% as the formulation number increased.
1.Characterization
1) Particle size and zeta-potential measurements
PBS buffer(pH 7.4) was used as diluted solution in order to quickly measure particle size and zeta potential. After each formulation was diluted 1000 times, it was measured at 25 degrees using a nanolaser particle size analyzer (ELSZ-1000, Otsuka Electronics Co, Osaka, Japan)
2) In vitro evaluation of O/W emulsion degradation
About 1 g of each formulation was put into the conical tube and the weight of the formulation was measured. The test was carried out using 20mL PBS buffer per one formulation. After 24 hours, the weight of each formulation remaining without PBS buffer was measured.
3) Rheology study
Each Rheological property was measured using Anton Paar MCR 702 MultiDrive Rheometer. In the dynamic viscoelastic measurement, the storage modulus (G'), loss modulus (G''), and complex viscosity (η*) were measured. Through thixotropic properties measurement, the properties of the emulsion were measured. The viscosity curve was measured in a range of shear rates between 0.1 and 100 s^-1.
2. In vivo evaluation of anti-adhesion effect
8 weeks male rats were used. The test was carried out in three groups that are control group, Guardix^®-sol group, No.4 group. After incision of each rat, the abdominal wall was scraped 200 times. 1mL of Guardix^®-sol group was applied to wounded site of Guardix^®-sol group rat. 1mL of No.4 group was applied to wounded site of No.4 group rat. 10 days after the incision operation, the abdominal cavity was opened again and checked.

Results: 1. Characterization
1) Particle size and zeta-potential measurements
The particle size of No.1 with 0.5% xanthan gum ratio and No.4 with 2% xanthan gum ratio were over 1000nm. In all formulations, the polydispersity index was less than 0.20 and zeta potential was measured as about -30mV.
2) In vitro evaluation of O/W emulsion degradation
No.4 swelled the most after the beginning of the degradation test and its weight increased rapidly. The higher ratio of xanthan gum the formulation had, the longer the formulation remained.
3) Rheology study
The storage modulus of each formulation was higher than the loss modulus. Viscosity of No.1 was higher than that of No.4, and the elasticity of No.4 was higher than that of No.1. In thixotropic properties measurement, the storage modulus of each formulation rapidly decreased when each formulation was applied with high shear. In viscosity measurement, the higher the shear rate, the lower the viscosity. The viscosity of No.4 had 5.7 times higher viscosity than that of No.1.
2.In vivo evaluation of anti-adhesion effect
When 10 days after surgery, the adhesion scores of the control group according to adhesion severity grading score and description were 4, 4, and 3. respectively. Those of Guardix^® -sol group were 2, 3, and 1. The No.4 group scored all 0.

Conclusion: Through rheology study, it was confirmed that all formulations have pseudoplastic behavior by xanthan gum, so they can come out smoothly from the syringes and recover the storage modulus. Because zeta-potential of all formulation is about -30 mV, the particles have a repulsion force and prevent agglomeration between particles. Through the degradation test result, it is considered that F4 has more hydrogen bonds than others because of high ratio of xanthan gum, so F4 decomposed slowly. According to in vivo evaluation results, F4 had the lowest adhesion score. Consequently, F4 has a better anti-adhesion effect compared to the existing Guardix®-sol.

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Figure 1. A) Formulations of O/W emulsions B) Particle sizes, polydispersity index (PI), and zeta potential of the emulsions C) Weight changes of No.1-4 emulsions remaining in PBS buffer (pH 7.4)


Figure 2. Rheology Study A) Dynamical oscillatory frequency sweep test curves of emulsions in xanthan gum concentrations ranging from 0.5% to 2.0%(w/w) B) Storage modulus, loss modulus, loss factor, and angle of the loss factor at angular frequency of 10 rad/s C) Thixotropy test curves of emulsions in xanthan gum concentrations ranging from 0.5% to 2.0%(w/w) at 10 rad/s angular frequency D) Viscoelasticity recovery rate of emulsions in xanthan gum concentrations ranging from 0.5% to 2.0%(w/w) at 10 rad/s angular frequency E) Viscosity test curves of emulsions in xanthan gum concentrations ranging from 0.5% to 2.0%(w/w)


Figure 3. A) Adhesion severity grading score and description B) Anti-adhesion effect of Guardix® -sol and No.4 emulsion C) In vivo evaluation of anti-adhesion effect on treated group and nontreated group (n=3) ; control, Guardix® -sol, No.4 emulsion in order from the top

Acknowledgements: This work was supported by the Mid-Career Researcher Program (No. NRF-2021R1A2C2008834), the Bio & Medical Technology Development Program (No. NRF-2022M3A9G8016257) and Basic Research Infrastructure Support Program (University-Centered Labs-2018R1A6A1A03023718) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science & ICT (MSIT).