Controlled drug delivery of ciprofloxacin from ultrasonic hydrogel

2.1 Material and devices

Sodium hydroxide, pure glycerin and acrylic acid were purchased from Merck (Germany). Acrylamide and methylene bis-acrylamide were purchased from Fluka (Germany). Analytical methylene bis-acrylamide was used without further purification. Acrylic acid was used after vacuum distillation, and double-distilled water was used as solvent. Table 1 shows the characteristics of the devices used in this project.

2.2 Ultrasonic hydrogel synthesis

Hydrogel synthesis was optimized using the classical method (15): 14 g 75% (w/w) glycerin as solvent and mixed monomer containing 0.75 g acryl amid and 0.75 g of acrylic acid neutralized 30% by NaOH were mixed with 0.1 g methylene bis-acrylamide cross-linker. Then the mixture was exposed to an ultrasonic probe with 80% (56 W) power and a pulse 8. At the end of the reaction, the obtained hydrogel was weighed and washed three times with water and ethyl alcohol and then dried in the oven 60°C for 8 h. The dry product was kept in a container away from light, heat and humidity.

2.3 Product analysis

The hydrogel samples were dried in vacuum at 60°C for 12 h to a constant weight. The dried hydrogels were analyzed in potassium bromide discs by Fourier transform infrared (FT-IR). In addition, UV-visible (UV-Vis) was used for the preparation of UV-Vis spectra. Scanning electron microscopy (SEM) was used to study the morphology of the hydrogels surfaces. Thermogravimetric analysis (TGA) was performed under nitrogen atmosphere.

2.4 The extinction coefficient measuring

To access the calibration curve, a 2.9×10⁻³m drug solution in water was prepared, and then solutions with concentration of 39, 79, 159, 238 and 318 mg/L were prepared via step dilutions of this solution. Ultraviolet absorption of these solutions was recorded in 271 nm. Extinction coefficient was calculated from the slope of the calibration curve (absorbance vs. concentration).

2.5 Swelling measurements

The method of tea bag was used to measure the speed of water absorption by hydrogel (16). This method is one of the most common and suitable criteria to measure the water absorption methods. Gel powder (0.2 g) was put in a tea bag and was suspended in 200 ml double-distilled water. Tea bag was hung after 3 h swelling of the hydrogel (10 min) to remove excess water, and the swelling capacity (g/g) was calculated using equation [1], where W_S and W_D are swollen and dry hydrogel, respectively.

2.6 Drugs loading in hydrogel

In order to investigate ciprofloxacin loading in hydrogel, its 2.1×10⁻³m solution was prepared, and 0.02 g of hydrogel was added to 20 ml of this solution in ambient temperature. Periodically, sampling was done from the upper part of the solution, and then it was analyzed with UV. After drug loading, the hydrogel containing the drug was filtered and washed with distilled water and was dried in an oven at 50°C for 8 h. Then dried hydrogel was washed with pure ethanol and was passed through filter, and the substrate solution was analyzed. Finally, the hydrogel was dried at room temperature and was ready for drug delivery. To study the effect of drug concentration, 0.02 g hydrogel was added to the samples mentioned in Section 2.4. Solutions were filtered after 30 min stirring and drug loading in the hydrogel, and the UV absorbance of each solution was measured.

2.7 Drug release from hydrogel

Due to the complexity of the human body simulation, the phosphate buffer in pH 7.4 was used to study drug delivery. To prepare the solution, 3.6 g of KH₂PO₄ and 5.79 g of Na₂HPO₄ were added to 1 L of volumetric flask with distilled water using the reference standard United States Postal. For drug delivery, the loaded hydrogel was put in a beaker containing 20 ml of buffer solution. The beaker was placed in a water bath at 37°C, the body temperature. Then the solution was analyzed at intervals of 30 min.

2.8 Effect of temperature and pH on drug release

To study the effect of the temperature, 20 ml of buffer solution was placed in a water bath with temperatures 5, 20, 37 and 45°C, respectively, and 0.02 g of gel containing the drug was added to each beaker. Then the solution was characterized by UV after each 15 min for 1 h. To study the effect of pH, some solutions at pH 2 and 8 (the stomach and intestines, respectively) and pH 10 were prepared. The solutions of sodium hydroxide and hydrochloric acid (0.1 m) were used to set the pH of the solution. Having adjusted the pH, 0.02 g of loaded hydrogel was added to each solution. The mixture was stirred each 15 min for 2 h, and the solution was characterized by ultraviolet.

2.9 Repeating experiments

Drug loading and release experiment were repeated in a different concentration of ciprofloxacin (2.9×10⁻³m) to confirm the obtained results and effects of the drug concentration.

3.1 Gel formation

Figure 2 shows the preparation steps of ultrasonic hydrogel from the formation to obtaining a dry sample. Method details are given in Section 2. After the gel formation, its swelling was studied as the most important property (17), (18), (19).

Figure 2:

Steps of processing of ultrasonic hydrogel preparation.

(A) synthesis, (B) filtering, (C) drying, (D) grinding and (E) swelling.

3.2 FT-IR and TG spectroscopy and structural analysis

The infrared spectrum was used to characterize and evaluate the functional groups. For this purpose, FT-IR spectra of the drug-loaded hydrogel, hydrogel and ciprofloxacin alone were recorded (Figure 3). In IR spectrum of ciprofloxacin, a broad peak in the area 3300–3500 cm⁻¹ related to the hydroxyl group O-H and an amino group NH in chain and the symmetric stretching vibration of methyl 2926 cm⁻¹ introduced a peak of moderate intensity and bending vibration in the area of 1451 cm⁻¹ CH₃ group, respectively. In addition, stretching vibration in the area of 1705–1725 cm⁻¹ belongs to the group C=O of ketones. Peak of the C-F vibrations was seen at 1000–1400 cm⁻¹. A peak with moderate intensity was seen in the 1600–1660 area, which is related to C=C double-bond vibration on the ring.

Figure 3:

Comparing of FT-IR spectra of drug absorbed on the hydrogel (A), hydrogel (B) and ciprofloxacin (C).

Comparison of IR absorptions shows that the frequency of 1624–1708 cm⁻¹ in loaded gel is related to two peaks, the first carbonyl group of drug C=O 11708 cm⁻¹ and the second C=C peak of the hydrogel.

Thermal gravimetric analysis diagram of prepared hydrogel has been shown in Figure 4. According to this diagram, thermal degradation starts with achieving temperature at 100°C and violent degradation at 600°C, and a significant reduction has occurred in the product mass. This destruction has continued up to 700°C with less intensity, and then no more changes were observed in product mass with increasing temperature. In addition, this figure shows the DTA charts (temperature vs. the symbols of reaction) in which we observed thermal analysis of two peaks, one minimum and another maximum. In this figure, the minimum peak shows dehydration process of components, as a result of an endothermic process, and the maximum peak can be the beginning of the crystallization and the formation of the first crystal, which is an exothermic process.

Figure 4:

TGA and DTA graph of hydrogel synthesized by ultrasound.

3.3 Effects of various factors on hydrogel swelling

In this section, the effects of several factors such as time, temperature and pH were studied on hydrogel swelling to be compared with the effects of these factors on the drug release process in the next part.

Table 2 and Figure 5 show the changes in hydrogel water absorption with time. As it can be seen, hydrogel swelling increases over time to about 60 min, and maximum water absorption is seen. It shows that the hydrogel synthesized by ultrasound has faster water absorption due to great pores.

Figure 5:

Hydrogel swelling changes over time.

Table 3 and Figure 6 show the water absorption changes of the hydrogel with temperature. As expected, hydrogel swelling has been reduced with increasing temperature. We can see maximum water absorption at 5°C and the lowest at 45°C; swelling lessens also at higher than 45°C. This behavior is due to a change in the lattice structure of the prepared hydrogels causing the flexibility of hydrogel structure to decrease with increasing temperature and water influence leading to decline gel swelling (20).

Figure 6:

The effect of temperature on the water absorbed by the hydrogel.

Due to the use of hydrogel in the pharmaceutical industry, it is important to study the behavior of its swelling with pH. Table 4 and Figure 7 show changes in the synthetic hydrogel swelling with pH. We can observe that swelling reaches its maximum (764.68) at pH 8 and then drops again. Ammonium groups (NH₃⁺) are formed in an acidic medium and the carboxyl ion (COO⁻) in basic environment. At pH 7 and above, water absorption and swelling increases with increasing ionization of carboxylic acid groups (COOH). However, at pH 10 and 4, swelling decreases due to charge overlap effect (3).

Figure 7:

Effect of pH on the water absorption of hydrogel (environments: acidic, basic and neutral).

3.4 Drug selection

In the selection of medical model to assess the loading and release, the intended drug should not react with hydrogel components or solvent. This interaction can be specified by shift in λ_max. This drug is a good case because no change was observed in the amount and location of λ_max over time. At first, its calibration curve was prepared at wavelength 271 nm (Figure 8). Extinction coefficient was obtained 0.008, equal to the slope of the curve (absorption vs. concentration). In addition, there was no interference between the drug and hydrogel spectra.

Figure 8:

Ciprofloxacin calibration curve (UV absorption vs. concentration).

3.5 Drug loading and the concentration effects

A 20-ml solution of ciprofloxacin (2.1×10⁻³m) was mixed with 0.02 g hydrogel at ambient temperature. Then hydrogel solution containing the drug was characterized by ultraviolet every 15 min, and its graph was plotted (Figure 9). It was observed that the highest absorption rate was at the beginning of drug loading on the hydrogel, and the drug was absorbed by the hydrogel and rate of absorption (drug concentration) reduced gradually over time (21), (22), (23), (24), (25). Drug absorption was complete approximately at 60 min, and then the absorption is fixed. This indicates that drug is loaded in the gel within a short time due to the large number of pores devised in the hydrogel.

Figure 9:

Plot of percent drug loading by hydrogel with time.

Ciprofloxacin has a maximum absorption in 271 nm, and absorption has a direct relationship with concentration (in less than 0.01 m), so we can use absorption changes in λ_max versus its concentration during loading in the hydrogel. Figure 10 shows the effect of drug concentration on the drug loading in the hydrogel. As we see, the amount of drug loaded in hydrogel increases with increasing drug concentrations, although the increase is not linear.

Figure 10:

The effect of drug concentration on the drug loading into hydrogel.

3.6 Drug release and factors affecting it

3.6.1 Drug release as a function of time

Drug release is conducted so that water penetrates the hydrogel network. Therefore, the release time of the drug is shortened by increasing the penetration rate of water into the hydrogel network. Accordingly, the drug releases from hydrogels, and its concentration increases in the buffer drastically over time (26), ( 27), ( 28). Figure 11 shows drug release changes from the hydrogel over time.

Figure 11:

Diagram of the hydrogel drug release over time.

3.6.2 Drug release as a function of temperature

In order to evaluate the effect of temperature according to Section 2.8, the magnitude of drug release was studied in different temperatures with time (Figure 12). Drug release is reduced as the temperature increases, and the least drug release is observed in the temperature of 45°C. As temperatures rises, the hydrogel network is undermined and its flexibility decreases. Thus, water penetration and hydrogel swelling reduces; thus, the lower buffer solution diffuses into hydrogel, and subsequently lower drug is released from the hydrogel. Low swelling and low release are desirable near the body temperature. On the other hand, gel occupies a smaller volume in intestine, and drug releases more slowly too and consequently has more opportunities to be absorbed. Also, in the residence time of the gel in gut (1 h or more), every loaded drug will be released and absorbed by blood.

Figure 12:

Drug release over time at different temperatures.

3.6.3 Drug release as a function of pH

Most of the drugs lose their structures upon entering the stomach (affect the gastric acid), and only a small amount of the drug absorbed through the intestines enters the blood. As a result, the effective amount of the drug needs to be increased as the drug enters the blood circulation, and this causes some side effects. pH sensitivity and biodegradability are very important for oral drugs. Because the swelling ratio of the drug-loaded hydrogels differs under the various pH values in the human digestive tract, the hydrogel can prevent drug release in the stomach (low pH), whereas the drug can be selectively released in the small intestine or colon (high pH) (29). Therefore, ciprofloxacin drug delivery is studied with time at pH 2 and 8, equivalent to the acidity of the human stomach and small intestines, respectively. As seen in Figure 13, drug release is greater at pH 8 rather than pH 2. This behavior is reasonable with the swelling pattern of hydrogel (Section 3.3) (3). Here, the highest drug release was observed at pH 8.

Figure 13:

Diagrams of drug release from the hydrogel at different pH levels.

Comparing the simultaneous effects of temperature and pH on drug release, we conclude that all the required conditions are preferred for the slow release of the drug in the gut, and this leads to the highest absorption of drug (30). It should be noted that the temperature is almost constant throughout the path of the drug, whereas the pH has many changes from the mouth to the anus.

3.7 Repeating loading and releasing experiments

In order to ensure the reproducibility of loading and delivery experiments, the entire process of loading and release of ciprofloxacin was repeated in another different concentration (2.9×10⁻³m). Other conditions were the same as mentioned before. The results were repeated here, similar to the previous concentration. The only difference was more absorption of drug due to more concentration. Data have not been shown here because of the high similarity and proximity to previous results.