Carbomer 940

Propranolol hydrochloride was purchased from Rouz Darou Pharmaceutical Co., Iran. Xanthan gum, guar gum and sodium alginate were obtained from Silverline Chemicals, India. Hydroxypropylmethyl cellulose 4000 (HPMC 4000) and sodium carboxymethyl cellulose (Na CMC) were from Shin-Estu Chemical Co., France. Carbomers including carbomer 934 (C934) and carbomer 940 (C940) were from BF Goodrich, Germany. Methanol, anhydrous citric acid, disodium hydrogen phosphate, propylene glycol and lactic acid were all purchased from Merck, Germany.
Preparation of propranolol hydrochloride gel formulationsDifferent classes of excipients usually incorporated in order to prepare vaginal gels include gelling agents, humectants, preservatives and vehicles (8 (link)). For this purpose, different concentrations of various mucoadhesive polymers including the natural polymers guar gum (in the range of 1-4% w/w), sodium alginate (in the range of 4-7% w/w) and xanthan gum (in the range of 2-5% w/w), and semi-synthetic polymers HPMC 4000 (in the range of 3-5% w/w) and Na CMC ( in the range of 4-7% w/w), as well as the synthetic polymers C934 and C940 both in the range of 0.5-2.0% w/w; were utilized to develop the gels. In order to formulate the mucoadhesive gels containing the drug, gelling agent was dispersed slowly in an aqueous-based solution containing propranolol hydrochloride (1.6% w/w, as the active ingredient), propylene glycol (5.0% w/w, as humectant) and sodium benzoate (0.25% w/w, as antimicrobial preservative), with the help of an overhead stirrer. The pH of the vagina is maintained by lactobacilli which produce sufficient lactic acid to acidify vaginal secretions to pH 3.5-4.5. The pH is important in terms of design and the efficacy of drug delivery systems (11 (link), 15 ). Hence, the pH of each formulation was adjusted to 4.0 (so as to be within the normal vaginal pH range) by the addition of lactic acid. Excipients are usually chosen from those materials which are deprived of therapeutic activity. Nonetheless, it is not always true; as it can sometimes be advantageous in the development of a pharmaceutical system (7 ). In this study, the main purpose of incorporating lactic acid into the formulations was its spermicidal activity (16 ). The composition of polymers within each of the gel formulations is given in Table 1. The prepared gel formulations were then tested on the basis of physical appearance, apparent viscosity, spreadability and strength of mucoadhesion. Then, four of these formulations were selected (named as chosen formulations) and underwent further examinations including determination of in-vitro drug release properties and drug release kinetic studies. Among these formulations, one formulation was selected as the final propranolol HCl gel formulation, which was then assessed in terms of complementary tests including propranolol HCl content within the gel as well as the duration of mucoadhesion.
Measurement of spreadability of gel formulationsThe area of spreadability of each propranolol HCl gel formuation, was determined using the following technique: five hundred milligrams (0.5 g) of the gel formulation was placed within a circle of 1 cm diameter, premarked on a glass plate, over which a second glass plate was placed. A weight of 500 g was allowed to rest on the upper glass plate for 5 min. The increase in the diameter due to spreading of the gel was noted (17 (link)) and then the spreading area was calculated using Equation 1, representing the area of a circle. This test was performed in triplicate and the data obtained expressed as mean ± standard deviation (SD).
A = π r² (Equation 1)
In the above equation, A is the area of the circle formed due to spreading of the gel (cm²), and r is the radius of the circle (cm).
Assessment of the mucoadhesive strengths of the gelsIn order to evaluate the mucoadhesive strength of the prepared propranolol HCl gel formulations, the apparatus shown in Figure 1 was used. This apparatus was principally similar to those described in previous studies (18 , 19 ). The upper stationary platform was linked to a balance, measuring the force needed to break contact between the gel and the mucosal membrane. The test cell was filled with pH 4.5 citrate-phosphate buffer, maintained at 37°C. Freshly removed sheep vaginal mucosa was used as the model mucosal membrane, and fixed in place over the two cylindrical platforms of the test apparatus and allowed to equilibrate in this solution for 2 min. Five hundred milligrams (0.5 g) of each gel formulation was then individually sandwiched between the two mucosa-covered platforms. Gels were kept in place for 5 min and then a constantly increasing force of 0.1 g/s was applied on the adhesive joint formed between the vaginal mucosa and the test gel, by gradually lowering the lower platform. This trend was continued until the contact between the test gel and the mucosa was broken and the maximum detachment force measured, was recorded. This force was taken as the strength of mucoadhesion of the test sample. Each experiment was run in triplicate, and results were expressed as mean ± SD.
Determination of in-vitro drug release profiles from the chosen propranolol HCl gel formulationsThe in-vitro release of propranolol HCl was determined from the chosen vaginal gel formulations using a dialysis tubing (MWCO of 12400 D; 99.99% retention, Sigma-Aldrich, USA) placed in the release medium under constant magnetic stirring. Five grams (5.0 g) of the gel formulations, were individually packed into sections of dialysis tubing (the length and the width of each section were 50 and 40 mm, respectively) with the ends being tightly fastened. The release medium was 200 mL of 0.1 M citrate-phosphate buffer (pH = 4.5). The medium was maintained at 37°C and stirred continuously at 100 rpm. Five mL (5.0 mL) aliquots of the release medium were withdrawn at predetermined time intervals and replaced by fresh citrate-phosphate buffer, to provide sink condition. Each withdrawn sample was further diluted with pH 4.5 citrate-phosphate buffer and it’s absorbance measured using uv-visible spectrophotometer (Shimadzu uv-visible 120A, Japan) at a λ _max of 289.2 nm. The absorbance was converted to drug concentration using the linear calibration curve constructed (Absorbance = 0.0196 Concentration (mg/L) – 0.0114; R² = 0.9995) and then cumulative percentage of propranolol HCl released was calculated with the help of a dilution factor. All measurements were performed in triplicate (n = 3).
In-vitro drug release kinetic studies of the chosen propranolol HCl gel formulationsIn order to study the release kinetics of the chosen propranolol HCl gel formulations, data obtained from in-vitro drug release studies were fitted into different kinetic mathematical models. These models were as follows: zero order (Equation 2), as cumulative percentage of drug released vs. time, first order (Equation 3), as Log cumulative percentage of drug remaining vs. time, and Higuchi’s model (Equation 4), as cumulative percentage of drug released vs. square root of time.
Q = Q₀ + K₀ t (Equation 2)
Where Q is the amount of drug released, Q₀ is the initial amount of the drug in the solution (it is usually zero), K₀ is the zero order rate constant expressed in units of concentration/time and t is the time.
LogC = LogC₀ – K₁t /2.303 (Equation 3)
Where C₀ is the initial concentration of the drug, K₁ is the first order release rate constant and t is the time.
Q_t = K_H t^1/2 (Equation 4)
Where Q_t is the amount of drug released in time t and K_H is the Higuchi’s model release rate constant reflecting the design variables of the system (20 (link)).
In order to evaluate the mechanism of drug release from the prpranolol HCl gel formulations, the first 60% drug release data were fitted in the Korsmeyer-Peppas model (Equation 5), as Log cumulative percentage of drug released vs. Log time.
M_t /M_∞ =Kt ⁿ (Equation 5)
Where M_t /M_∞ is the fraction of drug released at time t, K is the rate constant and n is the release exponent (20 (link), 21 ). The n value is used to characterize different release mechanisms, as given in Table 2 for cylindrical shaped matrices.
Determination of drug content within the final gel formulationFor determination of drug content within the final propranolol HCl gel formulation) of the gel was weighed in a 100 mL volumetric flask and then, 10.0 mL methanol was added to it (17 (link)). The content of the flask was stirred vigorously until the gel got completely dispersed to give a clear solution. The volume was adjusted to 100 mL with citrate-phosphate buffer pH=4.5. The obtained solution was diluted appropriately (dilution factor = 10) by the addition of pH 4.5 citrate-phosphate buffer and absorbance was measured in a uv-visible spectrophotometer (Shimadzu uv-visible 120A, Japan) at λ _max = 289.2 nm.
The absorbance was converted to drug concentration, using the linear calibration curve mentioned earlier. Then, the exact amount of the drug in the tested gel formulation was calculated with the help of dilution factor. This test was performed 3 times and the mean value ± SD was calculated.
Determination of duration of mucoadhesion of the final formulation The apparatus used for this study was based on that described in previous studies (19 , 22 ). The test apparatus (Figure 2) was composed of six upper and six lower cylindrical platforms within a clear jacketed perspex cell, filled with pH 4.5 citrate phosphate buffer. Freshly removed sheep vaginal mucosa (used as the model mucosal membrane) was mounted securely in place, mucosal side up-wards, on each of the platforms and allowed to equilibrate for 2 min. The test gel was then sandwiched between the two platforms and allowed to stand for 5 min. Next, through two pulley systems, a 7.0 g weight was applied on each upper platform (this weight was chosen through initial studies). As soon as the contact between the test gel and the mucosal surface broke, a small flap dropped onto a photocell detector, stopping the timer device (recording the elapsed time to 0.1 min) and measured the duration of mucoadhesion of the gel.
Statistical analysisData obtained from spreadability and strength of mucoadhesion of propranolol HCl gel formulations, were analyzed using the one way ANOVA and Tukey post-hoc test. Differences were considered to be significant at p < 0.05. The statistical package SPSS version 19.0 was used for data analysis.

To prepare EPR nanoemulgel, first hydrogel matrix was prepared by soaking (0.05, 0.1, and 0.2 g) carbomer 940 into water to form 10 g carbomer gel matrix. Then after, 1 g EPR nanoemulsion (EPR 0.05 g) was added to the gel matrix with slow continuous stirring. The final EPR nanoemulgel was obtained by adjusting pH to 6–7 with the addition of sodium hydroxide aqueous solution (Figure 1(B)). At the same time, the hydrogel incorporated with EPR emulsion was EPR emulgel.

The experimental materials included ethylene glycol (EG, AR) (Guangdong Guanghua Stock Science and Technology Co., Ltd., Shantou, China), polydimethylsiloxane (PDMS), carbomer 940 powder (Carbomer 940, AR), AA (>99%), AM (AR), N,N-methylenebisacrylamide (MBA, AR), 2-hydroxy-2-methylpropiophenone (1173, 97%), and sodium chloride (NaCl, AR, 99.5%) (Shanghai McLean Biochemical Technology Co., Ltd., Shanghai, China). The carbomer was dried at 60 °C for 24 h to remove moisture.

Carbomer 940 gel does not cause skin irritation and is the vehicle for the drug application in this article. First, 1 g of carbomer 940 powder (Meilunbio, Dalian, China) was mixed with 100 mL of ultrapure water and stirred for 5 min, and then the pH was adjusted to 7.0 with triethanolamine (Macklin, Shanghai, China). Subsequently, three different concentrations of LQ were added to formulate a 1% carbomer gel containing 0.5%, 1%, and 2% (w/v) LQ. The previous step requires stirring at 25 °C until a homogeneous and good-quality gel is formed [38 (link)]. What is more, cold paste (Haixu, Rizhao, China) is pre-made. LQ-CG-CP was prepared by uniformly applying LQ-containing carbomer gel to the cold paste. Its relevant characterization and in vitro drug release assays can be found in Figures S3 and S4 of the Supplementary Materials.

The carbomer 940 powder (400 mg) was weighed and put into a glass beaker with a magnetic stirrer, then treated with distilled water (20 mL) and stirred continuously standing to the full swelling at 50°C water bath. Sodium hydroxide (160 mg, 4 mmol) was dissolved in distilled water (4 mL). The sodium hydroxide solvation was added drop by drop to the swollen carbomer solution (pH = 5). The carbomer solution was gradually transparent and viscous and formed, and the final pH = 7 to obtain the hydrogel base, which was colorless and transparent.