Synthesis, Properties And Application Of Temperature-sensitive Biodegradable Vaginal Hydrogels

In the past few decades, vaginal gels as the carriers of various drugs have garneredmuch attention. Vaginal gels encapsulated a variety of anti-HIV drugs, contraceptive drugsand vaginal microbicides were widely investigated to promote the progress of femalephysiology, reproductive safety and the subdiscipline of biomedical materials.In the present paper, a series of copolymer, multiarm star-shapedpoly(D,L-lactic-co-glycolic acid)-b-methoxy poly(ethylene glycol)(PLGA-mPEG), weresynthesized via the arm-first method using linear PLGA-mPEG diblock as a arm andcarboxylation of polyols as a core. These multiarm block copolymers have the followingcharacteristics, namely, temperature-sensitive nature, amphiphilicity, biocompatibility,controlled degradability, multiarm star-shape on the structure and low viscosity of sol.Ethinyl estradiol (EE), gestodene (GSD) and indomethacin (IMC) were loaded into thecopolymer solutions using mixed method. This drug release system as a vaginal spraycould flow freely at room temperature and could not flow due to formation of gel at bodytemperature. This spray could be made into a small packaging, and after womenmenstruation a packaging was sprayed into the vagina at body temperature. Themesh-shaped gel formed quickly and attached to the vaginal wall. Estrogen, progesteroneand indomethacin in gel slowly released. At the same time, this spray degraded slowly anddrugs were released within about30days in the vagina and were re-sprayed.The main achievements were summarized as follows:1. A series of star-shaped block copolymers with various arm numbers,PLGA/mPEG block ratios, mPEG block length and LA/GA mol ratios were synthesizedusing arm-first method. And molecular structure, molecular weight and molecular weightdistribution of these star-shaped block copolymers were analyzed via Fourier transforminfrared spectrometer (FT-IR), gel permeation chromatography (GPC) and1H nuclearmagnetic resonance spectroscopy (1H NMR).2. The new method, in order to determining sol-gel transition process and transition temperatures, was established using static rheological curves. Combining with theinverting tube method, sol-gel transition behaviours of a series of star-shaped blockcopolymers with different arm numbers, PLGA/mPEG block ratios, mPEG block lengthand LA/GA mol ratios were investigated with changing temperature. Simultaneously,sol-gel transition behaviours of star-shaped block copolymers with adding hydrophilicmPEG homopolymer were also studied. PLGA/mPEG block mass ratio was the mostcritical factor to decide whether star-shaped block copolymers solutions had performanceof the sol-gel transition, and the ratios should be between2and3. The mPEG block lengthwas the most important factor to determine the sol-gel transition temperatures. The mPEGmolecular weight of550daltons was selected in order to gelation of copolymer solution atbody temperature. The sol-gel transition temperatures were coarsely tuned by selecting themolecular weight of mPEG block, and fine tuned by selecting PLGA/mPEG block ratios,arm numbers and LA/GA mol ratios.3. Formation and morphology of core-shell micelles were investigated via1H NMRand TEM. Star-shaped block copolymers in dilute solution with the concentration of1wt%self-assemble to form core-shell micelles with size of30-50nm. Micelle sizes anddistributions were further investigated using dynamic light scattering (DLS) with changingconcentrations and temperatures of dilute solution. Micellization and gelation mechanismswere subsequently discussed. The critical micelle concentrations (CMC) of copolymers indilute solution were studied via hydrophobic dye-UV spectroscopy method. The resultsshowed the critical gel concentration (CGC) was far greater than the critical micelleconcentration. Based on this, physical gelation process of star-shaped block copolymeraqueous solutions was discussed by us. Copolymer macromolecules in aqueous solutionsself-assembled and formed core-shell micelles, and then accumulated each other betweenthe micelles to cause the sol-gel transition. The hydrophobic interaction of PLGA blockwas considered as intrinsic driving force.4. With degradation time increased, changes in weight loss, molecular weight andcomposite of blocks of star-shaped copolymer hydrogels incubated in simulated vaginalfluid solution (SVF) in vitro and injected in neck of SD rats in vivo were investigated via weight method, GPC and1H NMR. At the same time, the differences between in vitrodegradation and in vivo degradation were compared. Weight and molecular weight ofstar-shaped copolymer hydrogels incubated in simulated vaginal fluid solution (SVF) invitro and injected in neck of SD rats in vivo decreased with decreasing LA/GA mol ratiosof star-shaped copolymer with the same molecular weight and block composite.Meanwhile, in vivo degradation rate of the hydrogel was quicker than in vitro degradation.The relative weight fractions of EG, GA and LA units were calculated using NMRend-group analysis with degradation time increased. Subsequently, degradation process ofstar-shaped copolymers in hydrogels was divided into three stages. In the first stage, esterbonds connected with the mPEG block were hydrolyzed mainly. In the second stage, esterbonds connected with the mPEG block and GA units of PLGA blocks were hydrolyzedmainly. In the last stage, ester bonds in LA units of PLGA blocks were hydrolyzed mainly.5. High performance liquid chromatography (HPLC) conditions simultaneouslydetecting drug content of ethinyl estradiol, gestodene and indomethacin were determined.Namely, optimal volume ratio of methanol and water as mixed mobile phase of HPLC was53/47, the retention time of indomethacin (IMC), gestodene (GSD) and ethinyl estradiol(EE) were11.18min,13.11min and15.57min, respectively. By examining three drugcontents of SVF solutions, cumulative release fraction of three drugs became bigger withdecreasing LA/GA mol ratios of copolymers, indicating that release of these drugs werecontrolled by degradation mechanism. By means of fitting of cumulative release curves ofstar-shaped copolymer hydrogels with various LA/GA mol ratios and drug-loadingcontents using Higuchi model, zero-order release model and first-order release model,good or bad order of the linear fitting relationship was as follows: Higuchi model>zero-order release model> first-order release model, indicating that drug releases of IMC,GSD and EE were suitable to Higuchi model.6. The star-shaped copolymers were basically biocompatible with low cellcytotoxicity due to high cell viability at all culture concentration and time by an MTTassay. Even though an acute inflammatory response occurred at the surrounding injectedsite of SD rats in early stage, the inflammatory response disappeared at late stage indicating that star-shaped copolymer hydrogels had a good biocompatibility. In addition,after spraying hydrogels with various drug dosages into vagina of SD rats, macroscopiccharacters and microstructure of organs were observed at various dosage groups andsprayed time. As to high dosage group, severe uterine edema, vaginal hyperemia, partiallysis necrosis of the liver cell, piecemeal necrosis of hepatocytes, and increasing spleenlymphocyte were found in SD rats. As to middle dosage group, uterine hyperemia was justobserved. As to low dosage group and control group, no significant pathological changeswere found. As a result, hydrogels loaded drugs with low dosage group is suitable to applyin female birth control spray.7. Interestingly, temperature-sensitivity of mixed copolymer aqueous solution wasfound using simply mixing two star-shaped copolymer solutions withouttemperature-sensitive property at different mixing ratios. Preparation method of the mixedgels, the performance of the micellization, sol-gel transition properties, in vitrodegradation, in vitro cytotoxicity and in vivo biocompatibility were preliminary studied.