Development of di(2‐ethylhexyl) phthalate‐containing thioglycolic acid immobilized chitosan mucoadhesive gel as an alternative hormone therapy for menopausal syndrome

Abstract Menopausal syndrome includes the symptoms that most women experience owing to hormone changes after menopause. Although hormone replacement therapy is a common treatment for menopausal syndrome, there are still many side effects and challenges hindering research. In this study, thioglycolic acid (TGA)‐immobilized chitosan mucoadhesive gel was synthesized by a new method of low concentration of 1,4‐butanediol diglycidyl ether (BDDE) would encapsulate di(2‐ethylhexyl) phthalate (DEHP) as an alternative hormone replacement therapy for menopausal syndrome. The efficacies of the DEHP‐containing TGA‐chitosan gel (CT‐D) were confirmed and evaluated by materials characterization and in vitro study. Results showed that CT‐D was not cytotoxic and had better mucoadhesive ability than chitosan. The animal model was constructed 1 month after bilateral ovariectomy in SD rats. CT‐D was administered intravaginally every 3 days. Bodyweight, wet weight of the uterus and vagina, vaginal smears, histology, blood element analysis, and serological analysis was used to assess the ability of the material to relieve menopausal syndrome. The results indicated that the combination of the sustained release of DEHP and mucoadhesive TGA‐immobilized chitosan allows the developed CT‐D to relieve the menopausal syndrome through low concentrations of DEHP, which falls in the safety level of the tolerable daily intake of DEHP.

women will drop from a normal or slightly high value to a low value (<30 pg/ml). 1 Follicle-stimulating hormones and the luteinizing hormone will also increase due to the lack of negative feedback from estrogen. [2][3][4] Owing to hormonal changes, most women experience uncomfortable physical and mental symptoms, which is called menopausal syndrome. Hot flashes are a major symptom of menopausal syndrome. It will give women a sensation of instantaneous temperature rise spreading through the whole body and may be combined with sweating, chills, heart palpitations, and anxiety. 5 Genitourinary syndrome is another common menopausal syndrome. The genitourinary tract of women undergoes a series of physiological and structural changes, including reduced vaginal rugae, decreased elasticity, loss of mature epithelial cells, and fibrosis of the epithelial tissue, which eventually causes vaginal atrophy. 6 Other studies have shown that reduced estrogen may also reduce the metabolic rate, which leads to weight gain in some women after menopause. 7 Hormone therapy is currently used to treat menopausal syndrome.
Estrogen and combined hormone therapy are the most effective ways to treat moderate to severe menopausal syndrome. However, menopausal hormone therapies that are in clinical use are still doubtful with regard to their risks and benefits in preventing chronic diseases. Clinical reports indicate that the use of these hormones increase the risk of coronary artery disease, breast cancer, stroke, and venous thrombosis. 8 Therefore, the current treatment recommendation is to use low-dose estrogen to improve menopausal syndrome.
In addition to the hormone therapy mentioned above, there have been many studies on alternative therapies. The use of selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, and selective estrogen receptor (ER) modulators can improve hot flashes, anxiety, and benefit the maturation of vaginal epithelial cells.
However, some side effects may occur, such as nausea, dry mouth, and constipation. 9,10 Other phytoestrogens, such as isoflavones, lignans, and coumestans as well as Chinese herbal medicines such as angelica, ginseng, ginkgo, and vitamin E are all potential alternative medicines, but their efficacies for menopausal syndrome vary for individuals. Some herbs are slightly toxic to the liver 11,12 ; therefore, hormone replacement therapy with lower side effects will provide a solution to the unmet needs of menopausal syndrome in clinical settings.
Di (2-ethylhexyl) phthalate (DEHP) is a common plasticizer used in the polyvinyl chloride industry. At high doses, DEHP causes reproductive and developmental toxicity in animals. 13 However, people are generally exposed to plasticizers daily with low concentrations of DEHP.
Approximately 70%-90% of DEHP is excreted after 36 h by a single oral dose. [14][15][16] More than 90% of the primary, secondary, and tertiary DEHP metabolites are rapidly excreted in the urine within 24 h postdose. 17 Some researchers have also shown that DEHP at a low concentration of 10 À3 mol/L could have estrogenic activities and stimulate the cell proliferation of human breast cancer Michigan Cancer Foundation-7 (MCF-7) cells in vitro. 18,19 In the vaginal delivery system, some polymers, such as hyaluronate, alginate, and methylcellulose 20,21 have been used in vagina atrophy; these polymers all have some limitations, for instance, leakage, short retention time, and lack of constant release in local delivery to the vagina. Conversely, the vaginal epithelium contains mucus layers, which can protect epithelial cells and prevent infection by microorganisms. 22,23 Because the mucus layers are composed of glycoproteins with cysteine-, glutathione-, and thioredoxin-rich domains, thiolated polymers are promising materials that may form disulfide bonds with the mucus layers. 24,25 In previous studies, the phytoestrogen genistein was encapsulated in a 1,4-butanediol diglycidyl ether (BDDE)-crosslinked thioglycolic acid (TGA)-immobilized chitosan gel.
The oxidation-reduction reaction in the vagina tract could cause the materials to dissociate from the mucosa to prevent side effects of the excess dose residue. 26 Presently, few studies have been conducted to evaluate DEHP to behave the estrogenic activities and be an alternative hormone replacement therapy for menopausal syndrome. In this study, we aimed to develop a DEHP-containing TGA-chitosan gel (CT-D). A relatively low concentration of BDDE was used to crosslink and immobilize TGA onto the polymer chain. The thiol group on the materials could react with mucin to form disulfide bonds to achieve a constant release of DEHP from CT-D for at least 3 days. We hypothesized that the combination of sustained-release DEHP and mucoadhesive chitosan gel would allow CT-D to relieve menopausal syndrome at a low concentration of DEHP, which falls in tolerable daily intake of DEHP. A low-dose DEHP and TGA-immobilized chitosan gel to keep DEHP-containing chitosan sustained release on the mucus layer would be a new idea for the long-term treatment of menopausal syndrome. The overall design is illustrated in Scheme 1.
The developed CT-D was characterized by Fourier transform infrared spectroscopy (FTIR), Ellman's assay, ninhydrin test, and energy-dispersive X-ray spectroscopy (EDS) to identify the functional groups and the thiol contents of TGA-immobilized chitosan.
High-performance liquid chromatography/tandem mass spec-

| FTIR analysis of CT
To synthesize the CT, BDDE was used as the crosslinker to react the epoxy groups on BDDE with the amines on chitosan and carboxylic acid on TGA. The functional groups of the synthesized CT were analyzed using FTIR spectrophotometry. The spectra were recorded in the wavelength range of 400-4000 cm À1 (Figure 1). The absorption band at 2496 cm À1 corresponds to the stretching vibration of the thiol (─SH) group. 27 The characteristic absorption band at 1220 cm À1 corresponds to the C SH stretching, which was not observed in chitosan. 27 The absorption bands at 1460 cm À1 and 910 cm À1 correspond to the C N C asymmetric stretching and N H wagging vibration of the secondary amine, respectively. 28 The bands at 2875 cm À1 and 2920 cm À1 were assigned to symmetric and asymmetric C H vibrations, respectively, which were mainly due to the crosslinked BDDE molecule. 29

| The thiol content of CT
The amount of free thiol groups on the synthesized CT was directly quantified by Ellman's assay, where the different concentration of cysteine was chosen as the standard. There were free thiols of approximately 0.58 ± 0.02 μmol/mg in CT, as shown in Figure 2a and summarized in Table 1.  Table 1.
The EDS analysis was used to determine the chemical composition of the CT, as shown in Figure 2c. Three random points were chosen for elemental analysis. The results showed that there was approximately 6.61% sulfur in the CT, as summarized in Table 2.
These results further proved that the TGA was successfully immobilized onto chitosan through BDDE crosslinking.

| In vitro cytotoxicity of CT-D
The in vitro cytotoxicity of the CT-D was tested using the WST-1 assay according to the International Organization for Standardization approximately 75% of cumulative DEHP was released on Day 3. The average 1 mg/ml/day of DEHP was released in the first 3 days, which is higher than the required concentration for estrogen activity. 18 This result indicated that controlled release of CT-D was achieved in this study, which was appropriate for the in vivo study.

| Mucoadhesive property of CT-D
Chitosan-FITC (CF) and chitosan-TGA-FITC (CTF) were used in the in vitro mucus adhesion test. As shown in Figure 5a, the materials were first attached to the mucus layer and then perfused with the culture medium. Fluorescence images were taken at different perfusion times.
The nuclei were stained with Hoechst 33342, shown as blue, where the FITC-labeled materials are shown in green. In the beginning, there were still many FITC-labeled materials in the medium, resulting in an obvious green background with the cells. Quantification of the FITC fluorescence image is shown in Figure 5b. The brightness at 0 h was set to 100%. As time progressed, the materials were washed away, whereas the CTF washed away slower than the CF. The results indicated that the thiols on the CTF could effectively conjugate to the mucus layer, thereby significantly enhancing the mucoadhesive property of the chitosan.

| DISCUSSION
After menopause, women experience systemic or local discomfort, such as hot flashes, night sweats, insomnia, vaginal atrophy, dryness, and itching due to a sharp drop in the concentration of estrogen in the body. 31 Currently, the prescription drugs used clinically are estradiol and its derivatives, but they may increase the risks of heart attack, stroke, and cancer due to endometrial hyperplasia. 32 To reduce side effects, local administration methods have been adopted, such as the use of estrogen cream, tablets, or rings, but their effects on menopausal syndrome are very limited and require frequent dosage. 33 In addition to the hormone therapy mentioned above, some people use water-, silicone-, or oil-based moisturizers and lubricants to improve vaginal moisturization and relieve the discomfort of vaginal atrophy caused by menopausal syndrome. 34 However, dyes, perfumes, and antibacterial agents in these products may be too irritating for the vagina. Their long-term effects are also insignificant and can only be used to relieve symptoms.
To address these unmet clinical needs, in this study, CT loaded with DEHP was designed for intravaginal delivery. There are many advantages of intravaginal administration, such as a considerable surface area, rich blood flow, high degree of permeability to most of the drugs, in addition to avoiding the first-pass effect of the drugs. [35][36][37] BDDE, a crosslinking agent widely used in hyaluronic acid, cellulose, and chitosan, [38][39][40] was used in this study to crosslink chitosan and simultaneously immobilize TGA onto the polymer chain. The thiol group content on CT was quantified by Ellman's assay, ninhydrin test, and EDS analysis, as presented in Figure 2 and summarized in Tables 1 and 2. The degree of grafting could be estimated by calculating the amount of the reduced free amino groups on chitosan and CT. In the ninhydrin test, about 0.54 μmol/mg of amino groups in chitosan were reacted with TGA through BDDE crosslinking, where the degree of grafting was approximately 10% ( Table 1). The grafted TGA on the chitosan chain provided CT-D with good mucoadhesive properties to the mucin ( Figure 5).
Polymer gels loaded with drugs have been shown to achieve prolonged, steady drug release. [41][42][43][44] In the mucoadhesive property experiment, both the chitosan and CT were retained on the mucus layer in the first 2 h ( Figure 5). When the medium was continuously perfused to mimic the vaginal environment, CT showed enhanced mucoadhesive properties compared to chitosan because CT could form covalent disulfide bonds with the mucus layer of the mucosal tissue to extend the retention time, which would enable the slow release of DEHP to avoid the self-cleaning mechanism of the vagina.
DEHP is a man-made chemical that is abundant in the environment. Numerous studies have shown that DEHP can interact with ERα and ERβ, acting on the endocrine system. 45,46 It has been shown that DEHP at a concentration of 10 À3 mol/L could behave as estrogen-like properties as 17β-estradiol. 18,19 The toxic dosage of DEHP and the metabolic mechanisms in the body are also fully understood. 14 The no-observed-adverse-effect-level (NOAEL) of DEHP was set at 4.8 mg/kg/day for multigenerational reproductive toxicity in rats. 17 Moreover, the tolerable daily intake value of DEHP for the parenteral routes is 0.6 mg/kg/day for humans. 47 increase cell debris. 31 When the rats were administered CT-D, the vaginal epithelium thickness recovered to 61% of the control group, and the mature cells recovered to approximately 37% of the control group (Figures 7 and 8). We speculated reasonably that the sustained release of DEHP would interact with the ERs. The ER activity in the vagina epithelium is important for maintaining the proper structural integrity of the vagina. 50 Hormone and growth factor signals, such as epidermal growth factor, keratinocyte growth factor, and insulin-like growth factor-1, would also be integrated by ER to mediate regulation of cell proliferation, vagina maturation, and thickness of the vagina epithelium. 51,52 Furthermore, other changes such as bodyweight and wet weight of the uterus and vagina also had a relative return to normalcy.
In the blood element analysis (Table S1) and serological analysis (- Table S2), most of the levels were in the normal range or consistent with the previous literature. 53,54 In addition, the materials can regularly excrete excess polymers and DEHP out of the body through the vagina's redox mechanism and cleaning mechanism. The results indicated that CT-D showed no systemic toxicity and could be a potential treatment for menopausal individuals.
In this study, the efficacy of the developed CT-D gel was determined by comparing it with that of commercial estrogen cream and ovariecto- Fetal bovine serum was purchased from Hyclone (Logan, UT, USA).

| Synthesis of TGA-immobilized chitosan (CT)
The CT was synthesized as follows: TGA (0.37 ml) was added to 50 ml of 2% (w/v) chitosan solution in 0.1 M acetic acid and stirred at room temperature for 1 h. The premixed solution was added to the crosslinker, 0.098 ml BDDE in 50 ml of isopropyl alcohol (IPA), and stirred at room temperature for 3 h. The resultant solution was dialyzed against deionized water using a dialysis membrane (MWCO: 12,000 Da) for 4 days. The solution was freeze-dried and stored at room temperature.

| Preparation of DEHP-containing CT gel (CT-D)
For the preparation of the DEHP-containing CT gel, DEHP was loaded into the CT gel as follows: A 3% (w/v) CT was prepared as in 0.1 M acetic acid.
DEHP was then added to the CT to achieve a concentration of 4 mg/ml.

| The analysis of FTIR
An FTIR spectrophotometer (Perkin Elmer, spotlight 200i) with an autoattenuated total reflectance (ATR) system was used to determine the functional groups of the CT. The detected wave number range was from 400 cm À1 to 4000 cm À1 . The number of scans was 16, and the resolution was 4 cm À1 .

| The analysis of Ellman's assay
Ellman's assay was used to evaluate the free thiol groups in the synthe-

| The analysis of ninhydrin test
Ninhydrin test was adopted to test the free amino group on the chitosan and CT and the procedure is briefly described in this section.

| Statistics
There were at least three replicates for each data point in this study and the values are expressed as "mean ± standard deviation." The calibration curve with a known concentration has a linear correlation coefficient greater than 0.99. The statistical difference of the experiment was tested using a one-way analysis of variance with multiple comparison tests. Differences were considered significant at a p-value of less than 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.

ACKNOWLEDGMENTS
This study was supported by the National Health Research Institutes Taiwan.

DATA AVAILABILITY STATEMENT
The data that supports the findings of this study are available in the supplementary material of this article ORCID Feng-Huei Lin https://orcid.org/0000-0002-2994-6671