The porous structure of the samples was characterized using transmission electron microscopy (JEOL JEM 2100 HR STEM – 200KV; point-resolution 0.23nm).

Determination of main physicochemical characteristics – particle size, index of polydispersity and zeta potential were performed using a Zetasizer Nano ZS (Malvern Instruments, UK). The samples were dispersed in distilled water and measured at a scattering angle of 90° and 25°. The measurements were made in triplicate.

The model drug substance was loaded into the pores of MCM-41 by using solvent impregnation method. Indomethacin (IND) was dissolved in 25 ml ethanol. Then, 100 mg mesoporous silica particles (MCM-41) preheated for 1h at 120 °С were added to indomethacin solution. After that, the mixture was incubated at predetermined temperatures under permanent stirring (180 rpm).

After 1, 3, 8, 15, 24 and 72 h, 50 ml of distilled water were added to the dispersion and ethanol was removed by vacuum distillation. The procedure continued with vacuum filtration (0.1 μm pore size), washing with distilled water and drying in a vacuum desiccator.

For determination of drug loading rate, 10 mg indomethacin-loaded particles were dispersed in 20 ml of methanol and stirred for 2 h at temperature 37 °C. Particles were then vacuum filtered using 0,1 µm membrane filters and re-dispersed in fresh methanol (20 ml). After a second filtration the supernatants were combine and the concentration of the indomethacin was determined by UV-vis spectrophotometry at a wavelength of 320 nm (Thermo Scientific Evolution 300). Standard curve (r > 0.998) was prepared in the concentration range 5–50 μg/ml.

Drug loaded particles (10 mg) were incubated into 100 ml simulated gastric fluid (pH = 1.2) and phosphate buffer (pH = 6.8) at 37 °C under continuous stirring (100 rpm). At appropriate time intervals, 3 ml samples were taken and centrifuged at 15,000 rpm for 15 min. Concentration of the released indomethacin was determined by UV-vis spectrophotometry following the procedure, described in “Drug loading determination” section.

Transmission electron microscopy was used for determination of the porous structure of non-loaded and indomethacin loaded particles (Fig. 1) in order to found potential changes in their mesostructures.

The unloaded MCM-41 particles (Fig. 1A) showed well-defined and homogeneous structure of the pores. The TEM images in Fig. 1B presented preserved porous structure after drug-loading procedures. The size of the particles that can be determined by the TEM technique correlated to DLS size-measurements.

Figure 1. 

TEM images of empty (picture A) and indomethacin-loaded (picture B) particles.

Measurement of particle size, polydispersity index (PI) and zeta-potential, obtained by DLS assay showed mean size 370 nm (PI 0.45) and a slight tendency for increase of particles size and polydispersity index after drug loading (Fig. 2).

Figure 2. 

DLS determination of main physicochemical characteristics (size, polydispersity index and zeta-potential) of empty (MCM-41) and indomethacin-loaded particles (MCM-41 IND).

Measurements of zeta potential showed specific for the mesoporous silica materials negative value. Changes were observed in the surface charge of the loaded particles. Decrease of zeta-potential was observed for indomethacin-loaded particles because of the negatively charged carboxylic groups in the structure of indomethacin. The strongly negative value of drug-loaded samples could be considered as an indicator for high physical stability of the particles.

Reaction temperatures used during drug loading procedure were selected according to the literature data. Three limit values were used – 21, 37, 60 °C. Fig. 3 contained the results of the conducted research.

Figure 3. 

Drug loading of MCM-41 after incubation at different temperatures.

As it could be seen, there was a proportional dependency between the reaction temperature and the degree of drug loading. Our results showed that the difference in loading between particles, incubated at 21 °C and these, incubated at 37 °C was significant. In our opinion, the optimal temperature for indomethacin loading should be 37 °C, as the increase in the temperature to 60 °C leaded to a very small increase in the loading but would result in the inability of some thermally labile drug substances to be included in mesoporous silica nanoparticles.

Incubation time was another factor that had a direct impact on the loading rate. For the aim of experiment six times intervals (1, 3, 8, 15, 24, 72 hours) were used. As it could be seen from Fig. 4, the loading increased with increasing reaction time, as this trend was more pronounced up to 24 hours. The loading rate was significantly increased, with 7% indomethacin loaded after one-hour incubation period, whereas after 24 hours incubation loading rate reached 36%. The additional three-fold increase in incubation time did not result in a significant increase in loading, so the optimum loading time should be set at 24 hours.

Figure 4. 

Drug loading degree (%) after different duration of the incubation stage.

The studies were conducted at three ratios between MCM-41 and indomethacin, namely 1:0.5, 1:1 and 1:2. Fig. 5 represented the results of drug loading as a function of carrier/drug ratio. As it could be seen, indomethacin loading increased with an increase in the concentration of the drug in the reaction mixture.

Figure 5. 

Drug loading rate (%) with using of different MCM-41/indomethacin ratios in the incubation stage.

In the conditions of a continuous increase of indomethacin concentration, the loading increased, albeit to a lesser extent. Our studies showed that ratios above 1:1 are inappropriate because of potential loss of drug substance during the loading process (Table 1).

The load process of unfunctionalized MCM-41was mainly based on a diffusion process. Since diffusion occurs until equilibrium was reached, addition of a non-solvent and solvent removal at the final stage of the loading would result in a change in equilibrium towards the nanoparticle channels, which would theoretically increase loading and reduce the loss of active substance. Our studies (Fig. 6), where ethanol was used as a solvent and distilled water as non-solvent, showed a very high increase in indomethacin loading using a non-solvent.

Figure 6. 

Drug loading degree (%) with and without using of non-solvent for indomethacin at the final stage of incubation process.

The influence of different drug loading rate on indomethacin release profiles was evaluated by in-vitro release tests. The tests were performed in two different media (pH 1.2 and pH 6.8) which are used to simulate the conditions in gastro-intestinal tract. Three different samples (respectively with 7, 15 and 35% drug loading) were used. The results are presented on Fig. 7.

Figure 7. 

Release profiles of samples with different drug loading in simulated gastric fluid (A, pH = 1.2) and phosphate buffer (B, pH = 6.8).

Our results showed that all samples had burst release in the first 10 min with a tendency for faster release with decrease of the drug loading at both pH media. The second phase (from 10^th min) showed different trend. The samples released loaded drug with different speed, depending on the loading rate. The particles with highest loading rate released indomethacin with slowest speed. Moreover, this tendency of indomethacin release in media with pH 6.8 was more pronounce (100% for 80 min).

This effect could be explained with the free spaces inside the mesoporous channels. Samples with low degree of drug loading had more free spaces in the channels. These spaces allowed more liquid to enter the pores which leaded to faster solubility of indomethacin. On the contrary, samples with higher drug loading possessed less free spaces which was premise for minimal contact between drug and gastro-intestinal fluids resulting in slower drug release.