The degradation of MSNs with varying surface-functionalization and dye/drug-loading was investigated using a dialysis setup in the aqueous buffer 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) at pH 7.2 and 37C, in order to mimic intracellular cytosolic conditions. The dissolution of silica was determined spectrophotometrically using the molybdenum blue method.14 The results of the degradation experiments, which were carried out over one week, are displayed in Figure 31. The surface chemistry (PEI-functionalization) as well as cargo (DiI/DAPT)-loading were found to clearly influence the degradation behavior of the investigated materials. All PEI-functionalized samples, regardless of dye/drug-loading, were found to initially degrade faster than the amino-functionalized particles (MSN). This phenomenon was thought to relate to the abundance of amino groups grafted onto the silica surface, which is known to boost silica degradation in neutral and alkaline conditions in the presence of water.212,213 The degradation profiles of the dye/drug-loaded further investigation of the
0 24 48 72 96 120 144 168 functionalization as wt% dissolved SiO2 over time.
Error bars are SD; n=3.
degradation samples with TEM (Figure 32) displayed a very intact outer spherical shape of the amino-functionalized MSNs while the pore structure had clearly been deformed, we proposed that these particles had primarily degraded from the inside by bulk erosion, which agrees with previously published data.441,442 The PEI-grafted unloaded sample (MSN-PEI), which displayed a more intact pore structure, was suggested to degrade by a combined bulk and surface erosion, since the absence of cargo would allow pore wetting and the surface amino groups would promote hydrolysis of Si-O-Si bonds, leading to surface erosion. Based on the same statements, the cargo-containing samples were suggested to degrade in a similar fashion as MSN-PEI with the addition of an initial retardation in dissolution rate due to the presence of hydrophobic cargo. The low asymptote value of 20-25 wt% silica dissolution for all investigated samples that was reached after one week of testing was thought to be related to limitations set by the experimental setup, including potential dissolution-reprecipitation behavior and the absence of solvent-exchange in the system. Naturally, at physiological conditions, the solvent-exchange of fluids would ultimately result in complete dissolution of the NPs.
Figure 32. TEM overview and single-particle images of MSN, PEI and MSN-PEI/DiI after dissolution and drug release testing for one week.
1.4 Drug release
The release of hydrophobic cargo (DiI/DAPT) from PEI-functionalized NPs was investigated in HEPES buffer at various solvents over one week, mimicking intracellular conditions in terms of temperature, pH, polarity, protein, enzyme or lipid content, presence of hydrophobic structures and finally also in live cancer cells. As can be seen in Figure 33, the release of cargo in pure aqueous buffer (HEPES) at pH 7.2 was very low both due to the limited water-solubility of the hydrophobic active agents and the presence of cargo that counteracted degradation-related release by preventing penetration of water molecules into the pore structure. A strong positive correlation between the release profiles and their corresponding carrier degradation profiles suggested that the release of poorly water-soluble cargo in a simple aqueous environment is strongly connected to the degradation of the carrier.
Figure 33. A) Dye and drug release profiles from MSN-PEI/DiI and MSN-PEI/DAPT particles in HEPES buffer (pH 7.2) at 37°C. Error bars are SD; n=3. The correlation between carrier degradation and cargo release is shown for B) DiI-loaded and C) DAPT-loaded particles.
Since only a small fraction of the total cargo amount was released at the employed conditions, further investigations of model drug (DiI) release in more complex media were performed (Figure 34). A decrease in pH to 4.8 of the release medium, which corresponds to the acidic environment found in late endosomes and lysosomes, where NPs typically end up after intracellular uptake, showed no signs of increasing the release rate. The dye release rather decreased, which reinforces the earlier statement that the release of hydrophobic agents in aqueous buffer is primarily dependent on carrier degradation, and agrees with knowledge of the pH-dependent solubility of silica.14,106 The presence of surfactants (concentration below CMC), enzymes or proteins in the release medium was also excluded as release-triggering factors.
Upon the addition of an organic apolar solvent to the release medium, an almost immediate release of adsorbed model drug was observed. Further investigations revealed that only the hydrophobic dye moved into the apolar phase, while the NPs resided in the aqueous phase. The desorption of model drug moreover reached a higher completion following an increased concentration (>CMC) of amphiphilic molecules or other hydrophobic components, such as cellular membranes and organelles, in the release medium. The drug-solubilizing capacity of the micellar and hydrophobic structures was clearly displayed and is possible to predict based on the high log P value (12.0) of DiI,443,444 which signals that DiI preferentially partitions into a more apolar substance.445 The strong mechanical agitation applied during experimental conditions should be taken into consideration when comparing the drug release profile to the drug release in actual intracellular conditions in live cancer cells. The lack of such a mechanical agitation at intracellular conditions is expected to result in a less pronounced contact between cargo molecules confined to the pores of the nanocarrier and surrounding hydrophobic structures, consequently resulting in a delayed release of cargo.
Figure 34. DiI release in different media plotted as % of dye released over time. Error bars are SD; n=3.