3.1. Fabrication and characterization of MSNs-CHI-FA
The aim of this experimental study was to design a pH-responsive and folic acid (FA) targeted drug delivery system using mesoporous silica nanoparticles (MSNs) coated with a chitosan (CHI) shell. The synthesis of the MSNs involved a sol-gel method with slight modifications based on previous reports, first time using chitooligosaccharide (CHO) as template. Subsequently, chitosan was linked to the surface of the MSNs using a silane coupling agent. To enhance the targeting capability towards cancer cells and minimize non-specific side effects, a self-targeting agent folate was conjugated to the chitosan thin film membrane of the nanoparticles. These surface chemical modifications resulted in the successful production of a pH-responsive MSNs-based drug delivery system with both targeting and control abilities.
The SEM and TEM image in Fig. 1a, 1b, and 1c shows that the blank MSNs exhibited a spherical and uniform morphology with ordered mesoporous structure, and the diameter of obtained MSNs determined was about 50 ± 5 nm. After modified by chitosan and FA, a distinct polymer shell layer covering the surface of MSNs with a slight increase in the average diameter was clearly observed in the TEM images (Fig. 1d), which was supposed to be the result of the self-assembly of chitosan on the surface of MSNs.
Figure 1e illustrates the zeta potential measurements of the mesoporous silica nanoparticles (MSNs) throughout the modification process. Initially, the zeta potential of the MSNs was determined to be 25.3 ± 0.2 mV. Upon the self-assembly of chitosan on the surface of the MSNs, the zeta potential increased to 18.2 ± 0.3 mV. This positive charge can be attributed to the abundance of amino groups present in chitosan. Subsequently, upon conjugation with FA, the zeta potential decreased to 12.4 ± 0.4 mV. This decrease can be attributed to the incorporation of negatively charged carboxyl groups from folate. Therefore, the observed changes in zeta potential confirm the step-by-step modification of chitosan and FA on the surface of the MSNs.
The FT-IR spectra of both MSNs and MSNs-CHI-FA exhibited transmission peaks at approximately 1084 cm− 1, 803 cm− 1, and 462 cm− 1, which were assigned to the characteristic stretching vibrations of the Si-O bond. Furthermore, the increased intensity of specific bands in the spectrum of MSNs-CHI can be attributed to the vibration of the C-H bond. In Fig. 1f, two new peaks at 1635 cm− 1 and 1540 cm− 1 were observed in the spectrum of MSNs-CHI-FA compared to MSNs. These peaks are attributed to the amide I and amide II infrared absorbance of the chitosan molecule, indicating the successful modification of chitosan on the surface of the MSNs. Subsequent conjugation with FA resulted in the appearance of new broad absorption peaks at 1608 cm− 1 and 1516 cm− 1, which can be attributed to the phenyl rings and amines of the FA molecules[23, 35]. This observation further supports the successful conjugation of FA to the MSNs-CHI-FA system.
Additionally, the thermal gravimetric analysis (TGA) depicted in Fig. 2 reveals a progressive decrease in weight with increasing temperature after each reaction. This observation provides further evidence for the successful modification at each step of the process.
The hexagonal array pore structure of the as-synthesized MSNs was analyzed using small-angle X-ray diffraction (XRD). Figure 3a displays the diffraction patterns of the samples, where distinct diffraction peaks corresponding to (100), (110), and (200) Bragg peaks were observed. This result further confirms the crystalline nature of MSNs. The reduced intensity of the XRD diffraction peaks in MSNs-CHI-FA compared to MSNs indicates the successful incorporation of chitosan and FA molecules onto the surface of MSNs. This change in intensity can be attributed to the modifications made to the MSNs structure. In Fig. 3b, it can be observed that both chitosan and FA modified MSNs, as well as unmodified MSNs, exhibit an amorphous structure.
The composition and elements of MSNs-CHI and MSNs-CHI-FA were analyzed by XPS. Figure 3c and 3d shows the XPS full-spectrum spectra of the samples. The shapes of the spectra are similar and the positions of the spectral lines are basically unchanged with slightly different intensities, which indicates that the constituent elements of the samples are the same, and there are only differences in the contents. All the samples are composed of Si, O, N and C. The spectral lines with binding energies of about 103 eV and 155 eV are those of Si2p and Si2s, respectively, and the lines with binding energies of about 25 eV and 52 eV are those of O2s and O1s, while those of about 265 eV correspond to the binding energies of C1s, and those of about 398 eV correspond to the binding energies of N1s[36–38]. The introduction of chitosan and folic acid did not significantly affect the composition of the SiO2 matrix.
The nitrogen adsorption-desorption isotherm of the nanoparticles exhibited a type-IV curve with capillary condensation, as depicted in Fig. 4a[39]. This curve indicates the presence of a typical mesoporous structure with a narrow pore size distribution. The hysteresis return line, observed in the range of P/P0 from 0.3 to 1.0, along with a small hysteresis loop, further supports the presence of mesopores. Upon calculation, the specific surface area of the particles was determined to be as high as 1443.7 m2g− 1, and the total pore volume was found to be 2.1712 cm3g− 1. The pore size distribution curves revealed that the mesoporous silica possessed a uniform mesopore size of approximately 2.5 nm, as shown in Fig. 4b.
3.2. Drug loading and in vitro drug release studies
Irinotecan (CPT-11), a semi-synthetic water-soluble anticancer drug, can be loaded into MSNs-CHI-FA through mesopores and is effective in minimizing the premature leakage of CPT-11 in the normal cellular environment after being capped by CHI. The loading efficiency of CPT-11 from MSNs-CHI-FA was determined by UV-vis absorption spectroscopy to be about 378 µg/mg, and its higher drug loading was attributed to the structural characteristics of the particles with higher pore volume.
To investigate the drug release behavior, we studied the CPT-11 drug release profile of MSNs-CHI-FA@CPT-11 particles in PBS solution (10 mM) release medium at different pH (7.4 and 6.0) (Fig. 5) Under neutral conditions (pH 7.4), the release of CPT-11 was very slow due to the formation of a primary membrane by chitosan on the surface of the particles. Only about 10.2% was released after 24 h. On the contrary, in a weakly acidic solution (pH 6.0, simulating the weakly acidic microenvironment of the tumor), the chitosan shell gradually dissolved and CPT-11 was released in large quantities, and the drug release efficiency increased to 40.8%.
3.3. In vitro cytotoxicity assay
We determined the in vitro cytotoxicity of MSNs, MSNs-CHI-FA, CPT-11, MSNs@CPT-11 and MSNs-CHI-FA@CPT-11 at different concentrations (1 ~ 100 µg/mL) by MTT assay. As shown in the Fig. 6a, the breast cancer cell viability was still higher than 85% when the particle concentration was up to 100 µg/mL, indicating that the cytotoxicity of MSNs and MSNs-CHI-FA was very low and almost negligible.
As for free CPT-11, the cytotoxicity of both MSNs-CPT-11 and MSNs-CHI-FA@CPT-11 showed dose-dependence (Fig. 6b). MSNs-CHI-FA@CPT-11 showed the strongest killing rate against tumor cells, which was attributed to the FA targeting effect with cancer cells, which promoted endocytosis increasing the intracellular accumulation of the drug and making more of CPT-11 acted into the nucleus and inhibited the cell activity.
3.4. Selective cellular uptake of MSNs-CHI-FA
In general, an ideal drug delivery system should possess the ability to specifically target and release cargo drugs to pathological or cancerous cells[40]. To address this requirement, we incorporated a folate (FA) modification on the surface of MSNs-CHI to confer targeting ability. To evaluate the efficacy of endocytosis, we conducted experiments using confocal laser scanning microscopy (CLSM) to examine the uptake of FA-modified MSNs-CHI by MDA-MB-231 cells that overexpress the FA receptor.
CLSM was used to observe the intracellular distribution of free CPT-11 after the particles were uptaken by breast cancer cells Fig. 7. CLSM images showed that more and stronger green fluorescence (CPT-11) could be observed in MSNs-CHI-FA@CPT-11-incubated cells compared to free CPT-11-treated cells. This further confirmed that MSNs-CHI-FA@CPT-11 improved cellular uptake efficiency and enhanced tumor drug delivery.