Optimization and Evaluation of the Quercus infectoria Galls Thermosensitive In Situ Gel for Rectal Delivery

Quercus infectoria galls (QIGs) have a long history of treating ulcerative colitis (UC). The aqueous extract of QIG has an anti-UC effect. However, QIG's enema is easy to leak, and the action time and dose of the drug cannot be controlled well. Thus, QIG is inconvenient to use. This study aims to screen and prepare an optimized thermosensitive in situ gel with slow release and retention. Taking the transition sol-gel temperature (Tsol-gel) as the investigation index, the Box-Behnken design response surface method (BBD-RSM) was used to optimize the dosages of Poloxamer 407 (P407), Poloxamer 188 (P188), and hydroxypropyl methyl cellulose (HPMC). Moreover, three formulations were selected, and the in vitro release rates were further optimized. The optimized rates of P407, P188, and HPMC were 24.07%, 1.22%, and 0.60%, respectively, and Tsol-gel was 32.8°C ± 0.4°C. The cumulative release of gallic acid in the gel conformed to the first-order kinetic equation, and gallic acid was released entirely within 24 h. In addition, the morphological and chemical characterization of thermosensitive in situ gel demonstrated that excipients did not affect the characteristic functional groups of QIG and that the surface of the QIG gel had a porous and loose structure. Rheological methods showed that the QIG thermosensitive in situ gel was fluid at low temperature and semisolid at gelation temperature. Therefore, the prepared gel was sensitive to temperature and had slow-release, local retention properties.


Introduction
Ulcerative colitis (UC), a nonspecific disease occurring in the colorectal mucosa, is a type of inflammatory bowel disease primarily characterized by ulcerative erosion [1,2]. e course of UC is long and often recurs [3,4]. e worldwide prevalence of UC ranges from 5.50 to 24.30/ 10000, whereas the prevalence of UC in mainland China is about 11.60/10000, which may be underestimated [5].
Quercus infectoria Olivier is a small tree or a shrub present in Greece, Asia Minor, Syria, and Iran. e tree grows galls due to infections by the Cynips gallaetinctoriae wasp [6]. Q. infectoria galls (QIGs, also known as Galla Turcica) have remarkable medicinal value and have been pharmacologically deciphered to have astringent, antidiabetic, antitremorine, local anesthetic, antipyretic, and the drug cannot be well controlled [11]. us, QIG is inconvenient to use. e rectal thermosensitive in situ gel has corresponding conditions at average ambient temperature and body temperature. Under the solution-gel transition characteristics, rapid gelation can be achieved at the administration location to achieve location adhesion, effectively prevent drug leakage, prolong retention time, improve the convenience of drug use, and realize location and continuous administration [12]. e thermosensitive in situ gel is expected to resolve the shortcomings of existing rectal dosage forms, effectively reduce the excretion of drugs after an enema, and form a drug reservoir on the surface of ulcers. e unique structure of the gel endows the drug with sustained-release performance to prolong the action time of the drug, improve the curative effect, reduce the discomfort of patients, and increase compliance. e most popular base of thermosensitive in situ gel is poloxamers (triblock copolymers of poly(oxyethylene)poly(oxypropylene)-poly(oxyethylene) [PEO-PPO-PEO]). Poloxamers comprise a central block of hydrophobic polypropylene oxide (PPO) surrounded on both sides by the blocks of hydrophilic polyethylene oxide (PEO) [13]. Poloxamers have good water solubility, surface activity, and reverse thermosensitive gelling properties and are safe. At a suitable concentration, the poloxamer system forms micelles in water with a dehydrated PPO chain as core and a hydrated PEO chain as shell [14]. With increasing temperature, the entanglement and stacking between micelles intensify, and the system changes from solution to solidification [15]. e gelling temperature (T sol-gel ) of the system is affected by the PEO/PPO ratio. erefore, T sol-gel of the system can be changed by adjusting the ratio of the poloxamer homolog [16]. Some mucoadhesive polymers, such as sodium alginate, hydroxypropyl methyl cellulose (HPMC), and carboxymethylcellulose sodium (CMC-Na), are commonly needed to increase the adhesion of the drug in the intestine [17]. erefore, in this experiment, the aqueous extract of QIG is used as primary drug to develop the thermosensitive in situ gel of QIG, improve the existing dosage forms of QIG, enrich the range of dosage forms combined with the reality of patients, flexibly choose the range of dosage forms, and provide a basis for expanding the scope of personalized treatment.

Preparation of QIG Extract.
Aqueous extraction was performed using a modified method reported in the literature [18]. QIG was pulverized into powder. About 200 g QIG passed into an 80-mesh sieve was collected and added with eightfold amount of distilled water. e mixture was soaked for 1 h and subjected to reheating reflux (Great Wall, China) three times for 0.5 h each time.
e extract was filtered using Whatman filter papers no. 4 (Cytiva, China, 20-25 μm), and filtrates were concentrated using a rotary evaporator (EYELA, China) at 55°C. e concentrated solution was collected in an evaporation plate, evaporated in the water bath (Jiangsu Jinyi Instrument, China) maintained at 80°C, dried, mashed, placed in a brown glass bottle, and stored in a refrigerator maintained at 4°C.

Preparation of QIG ermosensitive In Situ Gels.
e gel was prepared by the cold method after modification [19]. First, P188, P407, and HPMC were slowly added to the aqueous extract of QIG at a slow speed with magnetic stirrer (Tianjin Medical Instrument Factory, China) and continuously stirred for 5 min in a cold-water bath. en, mixtures were refrigerated at 4°C for 24 h for full swelling and to obtain a clear and well-dispersed QIG loaded gel solution.

Measurement of T sol-gel .
T sol-gel , also known as phase transition temperature, is the most important evaluation index of thermosensitive in situ gels. e test tube inversion and the agitator methods are commonly used to determine T sol-gel . However, when using the agitator method to determine gels, the accuracy of the results is challenging to ensure due to incomplete gel contact, uneven rotor rotation, and heating. erefore, the test tube inversion method with relatively high accuracy was selected to determine T sol-gel in this experiment.
T sol-gel of QIG thermosensitive in situ gel was determined by the inverted test tube method [20]. About 2 ml of different gel solutions under cold storage was placed in a glass tube with the same wall thickness and preheated in a water bath maintained at 15°C. e water bath surface was higher than the gel surface. e heating rate was 1°C·min −1 . e test tube was collected from the water bath every 20 s and tilted slightly to check the fluidity of the gel solution and presence of gelation until the gel was suspended in the test tube after the test tube was inverted. is temperature was T sol-gel of the gel solution.

Screening of Bioadhesive Materials.
In the mixed solution, P407 (24% [w/w]), P188 (1% [w/w]), HPMC, and CMC-Na were added to prepare solutions with concentration of 1%, and T sol-gel was measured by the method described in Section 2.4.
2.6. Single-Factor Test. First, P407 blank and drug-loaded gel solutions with different mass concentrations (18%-26%) were prepared, and T sol-gel was determined. Second, the concentration of P407 was fixed at 24%, and P188 (1%-5%) was added to prepare drug-loaded thermosensitive gel, and the gel temperature was determined. Finally, the concentrations of P407 and P188 were fixed at 24% and 2%, respectively, and the thermosensitive gel was prepared with 0.3%-1.5% HPMC. en, the gel temperature was determined.

Formulation Optimization Based on BBD-RSM.
e orthogonal test and response surface method (RSM) are commonly used in formulation process screening, and the RSM can obtain more accurate factor levels and has better experimental results than the orthogonal method [21]. erefore, in this experiment, the best formulation process was selected by the Box-Behnken design response surface method (BBD-RSM). BBD is one of the most adequate techniques for analyzing the data and optimizing the formula [22].

Design of Experiments.
Based on single-factor test results, the formulation of QIG thermosensitive in situ gel was optimized using the BBD-RSM. In the design phase, the contents of P407 (24%-26% [w/w], X 1 ), P188 (1%-4% [w/w], X 2 ), and HPMC (0.3%-1.2% [w/w], X 3 ) were used as the independent variables. T sol-gel was used as the dependent variable, with 3-factor and 3-level star point design experiment to optimize the thermosensitive gel formulation. e coded levels are −1, 0, and +1, and test factor levels and results are shown in Tables 1 and 2.

Analysis of Variance (ANOVA) and Significance Test.
Statistical analysis was performed using the Expert-Design ® 10.0.1.0 software. e relationship between independent and dependent variables was analyzed by the twice polynomial regression. e correlation coefficient (R 2 ) and confidence P values of the equations were used as criteria for model fitting.

Model Fitting.
e data in Table 3 were fitted by quadratic polynomial stepwise regression fitting by using the DesignExpert 10.0.1 software. One of the factors was fixed, and the two-dimensional contour map and threedimensional response surface map of the two other factors to T sol-gel were drawn.

Prediction and Validation of Optimal Formulations.
Given that the average human body temperature was 37°C, the gelation temperature was set to 33°C to ensure that the thermosensitive in situ gel was liquid at room temperature and could gel quickly at body temperature. T sol-gel of 33°C was used as the target temperature and substituted into the simulation equation. ree representative formulas in the optimized formula were selected, and three copies were prepared. e actual T sol-gel was measured, and the accuracy of the optimized method was verified and evaluated.

In Vitro Drug Release.
e in vitro release test was carried out in the TP-6 Franz diffusion cell [23] (XZH, China). e semipermeable membrane was clamped between the supply and receiving cells, and 17 ml PBS with pH 7.3 was injected into the receiving cell. After placing magnetic stirrer, the receiving pool drained out the air bubbles. About 1 ml formulation 1 gel was precisely added to the supply pool, and the gel was obtained by heating in an oven at 37°C for 2 min and immediately placed in a water bath with temperature of 37°C ± 0.5°C and rotational speed of 200 r·min −1 . About 600 μl was sampled at 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h, and then 600 ml 37°C PBS solution was immediately add. e released liquid was filtered by a 0.22 μm microporous membrane. e peak area of the device was measured by HPLC (Agilent1260 Infinity HPLC, Agilent Technology, USA). e cumulative percentage of drug release (%) was calculated, and the release curve was fitted and analyzed. e cumulative release was calculated in accordance with the following equations: where Q n is the cumulative release of the nth sampling point, C n is the concentration of the nth sampling point, V n is the volume of the dissolution medium, C i is the i-th sampling point concentrations, V i is the i-th sampling point sampling volume, and Q 0 is the content of gallic acid in the gel.    [24]. Specificity, linearity, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ) were evaluated. A certain concentration of the gallic acid standard solution (A), QIG sample solution (B), and blank sample solution (C) were injected into the HPLC system with the same operation. e chromatogram is shown in Figure 1. e peak of gallic acid was well separated and was not affected by excipients. For linearity studies, an eight-point calibration curve was prepared by diluting the standard working solution (160 μg·ml −1 ) with deionized water, and the range of this calibration curve was from 1.25 μg·ml −1 to 160 μg·ml −1 (Y = 15.089X − 33.649, r = 0.9992). Interday precision was realized using the same quality control samples (80 μg·ml −1 ) for six successive days, and the relative standard deviation (RSD) was 1.35%. Intraday precision was realized using the same quality control samples (80 μg·ml −1 ) in one day (RSD = 0.79%). Accuracy was determined by the addition of known amounts of GA standard drugs (at 50%, 100%, and 150% levels) to infusion samples in triplicate (RSD = 2.0%). LOD and LOQ were calculated using slope and Y-intercept (0.29 and 0.91 μg·ml −1 ).

Morphological Observation and Characterization of the QIG ermosensitive In Situ Gel with Scanning Electron Microscope (SEM).
e gel was freeze-dried using freeze dryer (LABCONCO, USA) for 24 h into powder, and the appropriate amount of gel powder was sprayed with gold, 15 kV accelerated voltage, and the microstructure and pore size of the gel surface were observed by SEM [25] (JOEL, Japan).

Fourier Transform Infrared (FTIR) Spectroscopy Characterization on QIG
ermosensitive In Situ Gel. FTIR spectra (Shimadzu, Japan) of formulation and excipients separately were obtained. About 2 mg QIG medicine, QIG water extract, blank thermosensitive gel, and QIG thermosensitive gel were separately placed in an agate mortar and added with 200 mg ground KBr. Mixtures were mixed evenly, placed into an ingot shaper, pressed into tablet form with 15 MPa (9t) for about 1 min, collected, and scanned 10 times. e resolution was 4 cm −1 , and the scanning range was 4000-500 cm −1 .

Sol Dynamics.
e tilting plate method was conducted as follows. e glass container was kept at a tilt angle of 45°at room temperature (25°C) for 5 min. en, 40 µl sol samples (blank sol and QIG-loaded sol) were dropped onto a glass plate. e time it took for each drop to slip down at a certain length was recorded. e sol kinetic curve was obtained by drawing the time-displacement curve according to these data [26].

Rheological Properties of QIG ermosensitive In Situ
Gel.
e viscoelastic properties of hydrogel networks were studied by placing a piece of thermosensitive gel between two plates through deformation-controlled (or stress-controlled) oscillatory experiment [27]. Taking the appropriate gels, carried out on a rheometer (Malvern, UK), table of   Evidence-Based Complementary and Alternative Medicine shear rates (TSR), shear stress ramp (SSR), and three-step test (TST) was tasted under rotational mode (CP4/ 40 stainless-steel parallel plates). en, in the oscillation mode (PU40 stainless-steel parallel plate), the sample was scanned by sequences amplitude sweep (AS), frequency scan (FS), and temperature scan (TS).

TSR of the QIG ermosensitive In Situ Gel.
e TSR test of QIG thermosensitive in situ gel was performed at 4°C, 25°C, and 37°C to determine the fluid type of the sample in rotation mode. e shear rate (c) was set to 0.1-100 s −1 , and the relationship among c, shear stress (σ), and viscosity (η) was determined.

SSR of the QIG ermosensitive In Situ Gel.
e SSR test of QIG thermosensitive in situ gel was performed at 4°C, 25°C, and 37°C to determine the gel's retention. e relationship between σ and η was determined by setting σ and time to 200 Pa and 100 s, separately.

TST of the QIG
ermosensitive In Situ Gel. Shear thinning materials may have thixotropy, whereas thixotropic materials are continuously undergoing shear thinning. e QIG thermosensitive in situ gel was tested by TST at 4°C, 25°C, and 37°C to determine whether the samples had thixotropy. e relationship between time and viscosity was observed by setting the shear rate and time as 0.1 s −1 and 30 s, 100 s −1 and 600 s, and 0.1 s −1 and 30 s, respectively.

AS of the QIG ermosensitive In Situ Gel.
e QIG thermosensitive in situ gel was tested by AS at 4°C, 25°C, and 37°C to determine the maximum deformation that the internal three-dimensional reticular structure of the gel could bear when destroyed. Measurement conditions were strain amplitude (c * ) of 0.1%-100% and frequency (f) of 1 Hz. Associations between c * and storage modulus (G ′ ), loss modulus (G ″ ), and phase angle (δ) were determined.

FS of the QIG
ermosensitive In Situ Gel. Frequency scanning was designed to describe the timedependent characteristics of the sample in the range of nondestructive deformation. e temperature was controlled at 4°C, 25°C, and 37°C; the strain was 1%; and the frequency was set to 0.1-10 Hz. e frequency of the QIG thermosensitive in situ gel was measured in oscillation mode, and the relationships between f and G ′ , G ″ , and δ were determined.

TS of the QIG
ermosensitive In Situ Gel. e frequency was set at 1 Hz, and the strain was 1%. e sample was heated at a rate of 5°C·min −1 , and the temperature range was 5°C-50°C. Changes in G ′ , G ″ , complex η (η * ), and δ with temperature were measured.
2.14. In Vivo Images of the ermosensitive Gel. NIR fluorescence imaging images were obtained and processed with the IVIS Spectrum (Perkin Elmer, USA) to know the residence time of gels in the rectum. Ten female KM mice (12 weeks old) purchased from the Animal Center of Xinjiang Medical University (Experimental Animal Production License no. SCXK [New] 2018-0002) were used. Mice were randomly divided into the thermosensitive gel and common enema groups. Before the experiment, mice were fasted for 24 h and drank regular water. First, 100 μl sample with mixed IR820 was removed with a pipette and gently inserted into the rectum of the mouse at depth of about 0.5 cm to push the QIG thermosensitive gel and QIG enema. Mice were anesthetized by the intraperitoneal injection of 10% pentobarbital sodium (0.05 ml·g −1 ) to make mice lie on their back and expose their abdomen. en, the mice were exposed at 0, 1, 2, 3, 4, 6, 8, and 24 h under the same exposure intensity, and fluorescence signal images were collected.

Screening of Bioadhesive Materials.
e effect of HPMC on T sol-gel was within the range of human physiological temperature. us, HPMC was chosen as adhesive (Table 4).

Single-Factor Test.
e single-factor test was carried out to reduce the proportion of each auxiliary material. As shown in Table 3, T sol-gel decreased with increasing P407 concentration. T sol-gel of P407 increased with the accession of QIG extract compared with the blank gel. When the dosage of P407 of QIG-loaded gel was less than 24%, the solution could not be gelled at 37°C. us, the range of optimal dosage of P407 was 24%-26%. By comparison, T sol-gel increased with the addition of P188 and HPMC. Considering the matching with the dosage of P407, the dosage of P188 was 1%-4%. However, HPMC increased slightly but had a minor effect. Considering the matching with P407 and P188, the optimization range of HPMC dosage was 0.3%-1.2%.

ANOVA and Significance
Test. ANOVA was used to analyze experimental data (Table 5). e model's P < 0.0001, indicating there is a pretty significant difference. R 2 was 0.9891, and the adjusted R 2 was 0.9750, pointing that this model could illustrate the change of 98.9% response value and reflect the relationship among P407 (X 1 ), P188 (X 2 ), HPMC (X 3 ), and T sol-gel (Y). e lack-of-fit P was 0.8975 > 0.05, which was not significant. us, no misfit factor was observed. X 1 , X 2 , and X 3 had significant effects on T sol-gel .

Model Fitting.
e result of the fitting equation was as follows: T sol-gel � 31.58 -3.00X 1 + 1.48X 2 + 0.73X 3 + 0.000 2 + 0.100 3 − 0.050 3 + 0.035X 1 2 + 0.085X 2 2 − 0.19X 3 2 . e twodimensional contour map and the three-dimensional effect map of the experimental results were drawn using the DesignExpert 8.0.6 software (Figure 2). Table 6 shows that the deviation between the measured and predicted values of the gel temperature of each formulation was less than 2%.

Prediction and Validation of Optimal Formulations.
is result indicated that the fitting equation had good predictability.
At room temperature, the QIG thermosensitive in situ gel is a yellow uniformly dispersed solution with good fluidity. e gel is yellow and semisolid above the phase transition temperature (T sol-gel ≥ 32.8°C) (Figure 3). Figure 4, the release curves of the 3 formulations did not coincide and were fitted by zero-order kinetics, Ritger-Peppas, Higuchi, and first-order kinetic distribution model (OriginPro 8.0 software). e goodness-of-fit was measured by parameters, such as correction determination coefficient (R 2 ) and analysis of variance (P). R 2 is closer to 1, and P < 0.05 indicates that the fitting is statistically significant. As shown in Table 7, the first-order kinetic distribution model was the best to fit the release curve of each formulation. T 50 indicated the time taken to release 50% (Table 8) and was more than 3 h, showing apparent sustained-release performance. Given that F1 was entirely released within 24 h and the shared key quality attributes could be achieved using the least number of excipients, F1 was the optimal formulation. Figure 5, the blank gel (A) had multiple round holes, a loose lamellar structure, void penetration, uniform internal structure, and porous connected mesh. e QIG in the QIGloaded gel (B) was uniformly dispersed in the lamellar gel structure, and gel pores were filled, indicating that the aqueous QIG extraction could uniformly disperse and fill in   the mesh and pores of the gel base surfaces. ese physical changes led to the strength of the gel and determine the release law of the QIG.

FTIR Spectroscopy of the QIG ermosensitive In Situ Gel.
e FTIR spectra of the raw medicinal powder, aqueous extract, QIG-loaded gel, and blank gel are shown in Figure 6. e spectrum, which included multiple characteristic absorption peaks, was complex. A series of high-intensity characteristic absorption was found in the 1710 to 1448 cm −1 region. e peak at 1708 cm −1 was related to the stretching vibration of C�O in the gallic phthalein group [28]. Given the stretching vibration of the C�C group and the deformation and stretching vibrations of the aromatic  Evidence-Based Complementary and Alternative Medicine 7 skeleton and C-H in the plane, a characteristic peak appeared at 1612-1530 and 1448 cm −1 [28], and the absorption bands at 1325 and 1201 cm −1 were related to the symmetrical in-plane bending vibrations of CH 3 and CH 2 [29]. erefore, excipients did not affect the characteristic functional groups of the API, ensuring the maximum efficacy of the API.

Sol Dynamics.
A certain concentration of P407 had reversible temperature-sensitive gelling properties. P407 was liquid at low temperature, became semisolid when the system rose to a certain temperature, and changed back to liquid when the temperature decreased [30].
e main mechanism of gelation in P407 solution was micelle stacking and entanglement. P407 formed a spherical micelle with hydrophobic PPO block as core and wrapped in   hydrophilic PEO shell. With increasing temperature, a high polymer concentration resulted in high number of micelles and probability of contact and entanglement. us, the gelation temperature was dependent on concentration [31,32], as shown in Figure 7.
Based on the above theory, the QIG-loaded gel took more time to complete the same distance of sliding than blank gels. A plausible explanation was that, with the addition of aqueous QIG extract, the water molecules on the PEO blocks became tightly entangled, resulting in difficulty of removing water molecules from PEO blocks. is phenomenon led to a slow kinetic rate of the sol. e addition of the QIG to the sol had a significant effect on the kinetics of the sol. e addition of QIG decreased the kinetic rate of sol, improved the adhesion, and then increased the QIG release rate in the gel (Figure 8).

TSR of the QIG ermosensitive In Situ
Gel. e behavior of the sample in different processes was simulated by c. A very low c (∼0.001 s −1 ) was used to evaluate the stability and quality of the sample, intermediate c (∼10 s −1 ) was used to evaluate pumpability and paintability, and low c (∼1 s −1 ) was used to evaluate the fluidity. High c (∼100 s −1 ) was used to determine whether the sample was too thick to spread. e three general flow behaviors were Newtonian, pseudoplastic, and dilatant plastic fluids.
When the c table was at 4°C and 25°C, σ of the QIG thermosensitive in situ gel increased linearly with increasing c, whereas η remained unchanged. is result showed that the gel had the properties of a Newtonian fluid. However, at 37°C, σ and c of the sample tended to be stable, and sample η decreased with increasing c, indicating a pseudoplastic fluid. e QIG thermosensitive in situ gel was inferred to show the state of pseudoplastic fluid after in vivo administration. Results are shown in Figure 9.

SSR of the QIG ermosensitive In Situ Gel.
e QIG thermosensitive in situ gel had no peak value at 4°C and 25°C, indicating no yield stress. is finding was consistent  Evidence-Based Complementary and Alternative Medicine with the results of TSR, and the sample cannot flow. At 37°C, the curve increased first and then decreased, and the peak value was evident. Figure 10 shows that the gel structure of the sample was formed and that the gel needed particular stress to flow. A special retention force in the drug administration site was preliminarily predicted.

TST of the QIG
ermosensitive In Situ Gel. Figure 11 shows the change in η with c tending to be a straight line, indicating that the sample is Newtonian fluid at 4°C and 25°C. However, the sample has a small shear thinning process at 37°C, it can recover instantly after the high shear process, and the relationship between time and  viscosity tends to be linear. erefore, the gel has no thixotropy and is not time-dependent.

AS of the QIG ermosensitive In Situ Gel.
Results are shown in Figure 12. At 4°C and 25°C, G ″ > G ′ , and δ > 45°i ndicated that the lost part of the sample was larger than the stored part having fluid characteristics, which is a viscoelastic liquid, and also G ′ G ″ does not change with the increase of c * . However, at 37°C, c * < 1%, G ′ > G ″ , and δ < 45°indicated that the elasticity of the sample was dominant, representing a gel structure. When G ′ � G ″ the gel structure is destroyed, G ′ < G ″ and δ > 45°indicated the viscosity of the sample was dominant, and it shows up fluid properties. erefore, all dynamic oscillation experiments should control c * within 1%.

FS of the QIG ermosensitive In Situ
Gel. At 4°C and 25°C, G ″ was always larger than G ′ , and δ was much larger than 45°, showing evident viscous fluid characteristics. At 37°C, samples G ′ and G ″ evidently increased by about four orders of magnitude. G ′ > G ″ and δ < 45°showed evident elastic properties, and no significant frequency dependence  Evidence-Based Complementary and Alternative Medicine was observed, indicating that the sample always had a stable crosslinking structure ( Figure 13).
3.8.6. TS of the QIG ermosensitive In Situ Gel. Figure 14 shows that, with increasing temperature, G ″ and η * increased, whereas δ decreased. At about 32°C, G ′ and G ″ intersected, and the intersection point was its phase transition temperature (T sol-gel ). Before T sol-gel , sample G ″ > G ′ , η * was small, and δ > 45°. ese results indicated that the sample had G ″ and low η, showing prominent fluid properties. It gradually increased G ′ > G ″ , η * increased sharply, and δ < 45°after T sol-gel . is result indicated that the gel structure was formed, and the gel structure G ″ , η * , and δ   tended to be flat, which proved that the viscoelasticity of the gel tended to be stable.

3.9.
In Vivo Images of the ermosensitive Gel. e duration of the material after enema in vivo should be determined for controlled drug release. e distribution and retention of QIG in mice in the ordinary and thermosensitive gel enema groups could be observed through fluorescence imaging. In Figure 15, partial leakage could be seen with naked eyes after administration in the ordinary enema group. Fluorescence signals could be observed on the back paw, fur, and tail of mice. In the thermosensitive gel enema group, QIG extended and gelled from rectum to colon, and the fluorescence signal could still be observed at 12 h. In the ordinary enema group, QIG was almost distributed all over the body at 1 h. is result indicated that the temperature-sensitive gel could prolong the local retention time of new indocyanine green.

Conclusions
e QIG thermosensitive in situ gel was prepared by the cold method. First, the preparation process used T sol-gel as index. e number of excipients was investigated and selected by the single-factor test. Second, on the basis of the single-factor experiment, the dosages of P407, P188, and HPMC were optimized by BBD-RSM. In accordance with the optimized screening results of the number of excipients and by using the drug release performance in vitro as index, the optimal preparation process of QIG thermosensitive in situ gel was determined. e best preparation results were as follows: P407 (24.07% [w/w]), P188 (1.22% [w/w]), and P188 (0.60% [w/w]), and T sol-gel was 32.8°C ± 0.4°C. e in vitro cumulative release curve of QIG thermosensitive gel followed the first-order kinetics equation: Q � 119.68 × (1 − e −0.09t ) (r � 0.9968) and it was completely released within 24 h. SEM results indicated that the blank gel had a porous connected three-dimensional network structure. QIG was uniformly dispersed in the gel lamellar structure, and gel pores were filled. is change might increase the strength of the gel. FTIR analysis verified that excipients had no effect on the characteristic peak of QIG, and rheological studies confirmed that the QIG thermosensitive in situ gel had Newtonian fluid properties, no yield value and thixotropy at 4°C and 25°C, pseudoplastic fluid property, and yield value without thixotropy at 37°C. In vivo imaging showed that the fluorescence signal could still be observed in the QIG temperature-sensitive in situ gel group at 12 h and that IR820 was almost all over the body at 1 h in the ordinary enema group, confirming that the QIG thermosensitive in situ gel could prolong the local retention time of IR820.
In summary, the prepared QIG thermosensitive in situ gel has the functions of temperature sensitivity, sustained release, strong spreadability, and high retention. In addition, the stability, quality standard, rectal mucosal irritation, pharmacodynamics, and pharmacokinetics of the QIG thermosensitive in situ experimental gel test should be further studied to improve the preclinical research.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.  Figure 15: NIR fluorescence of KM mice administered with IR820-loaded QIG thermosensitive in situ gel and IR820-loaded ordinary enema at different time points.