Autonomous in vitro anticancer drug release from mesoporous silica nanoparticles by pH-sensitive nanovalves

Abstract

Mesoporous silica nanoparticles (MSNP) have proven to be an extremely effective solid support for controlled drug delivery on account of the fact that their surfaces can be easily functionalized in order to control the nanopore openings. We have described recently a series of mechanized silica nanoparticles, which, under abiotic conditions, are capable of delivering cargo molecules employing a series of nanovalves. The key question for these systems has now become whether they can be adapted for biological use through controlled nanovalve opening in cells. Herein, we report a novel MSNP delivery system capable of drug delivery based on the function of beta-cyclodextrin (beta-CD) nanovalves that are responsive to the endosomal acidification conditions in human differentiated myeloid (THP-1) and squamous carcinoma (KB-31) cell lines. Furthermore, we demonstrate how to optimize the surface functionalization of the MSNP so as to provide a platform for the effective and rapid doxorubicin release to the nuclei of KB-31 cells.

Figure 1

A graphical representation of the pH responsive MSNP nanovalve. (a) Synthesis of the stalk, loading of the cargo, capping of the pore, and release of the cap under acidic conditions. Based on our calculations, the maximum number of stalks per nanopore is 6, and the maximum number of fully assembled nanovalves per nanopore is 4. The average nanopore diameter of the MSNP is around 2.2 nm, and the periphery diameter of the secondary side of β-cyclodextrin is ~1.5 nm. Thus, for a cargo with diameter >0.7 nm, a single nanovalve should be adequate to achieve effective pH-modulated release. (b) Details of the protonation of the stalk and release of the β-cyclodextrin. (c) TEM image of capped MSNP. The scale bar is 100 nm.

Figure 2

Release profiles of cargo molecules and the cyclodextrin. (a) Fluorescence intensity plots for the release of Hoechst dye, doxorubicin, and the pyrene-labeled cyclodextrin cap released from MSNP. (b) Release profiles of doxorubicin from ammonium-modified (7.5%, w/w) nanoparticles showing the faster and larger response compared to the unmodified MSNP (Figure 2a).

Figure 3

Cellular uptake and lysosomal pH measurements in THP-1 and KB-31 cells. (a) Confocal microscopy images showing FITC-labeled MSNP uptake into the LAMP-1⁺ compartment in THP-1 and KB-31 cells. The yellow spots in the merged image show the colocalization of the nanovalve MSNP with LAMP-1 positive compartment. The colocalization ratio, as determined by Imaging J software, indicates >80% colocalization of the green-labeled nanoparticles with the red-labeled lysosomes. (b) Measurement of lysosomal pH in THP-1 and KB-31 cells prior to and after NH₄Cl treatment (*p <0.05). The dashed line indicates that a threshold pH (6.0) is required for nanovalve opening. Since NH₄Cl treatment elevates the pH to above this threshold, it eliminates the microenvironment that is required for cargo release.

Figure 4

Confocal images of THP-1 and KB-31 cells incubated with MSNP containing Hoechst dye and doxorubicin drug for the indicated times. (a) Hoechst-loaded, FITC-labeled MSNP are efficiently taken up in THP-1 cells. In early time points (1 and 3 h), Hoechst was retained in nanoparticles localized in the perinuclear region. The gradual disappearance of blue dots in perinuclear region and gradual increase in nuclear staining indicate the release of Hoechst dye from the nanoparticle to the nucleus. By 36 h, most of dye was released. NH₄Cl effectively prevented Hoechst release. (b) Quantitative analysis of the nuclear Hoechst fluorescence signal was determined by Image J software in cells with or without NH₄Cl treatment. (c) KB-31 cancer cells effectively endocytosed the doxorubicin-loaded FITC-MSNP at 3 h. This action is followed by doxorubicin release to the nucleus, induction of cytotoxicity and appearance of apoptotic bodies by 60 h (arrow in Figure 4c), followed by nuclear fragmentation after 80 h. However, with NH₄Cl treatment, most of the doxorubicin was confined to the nanoparticles and hence there was no observable cell death. (d) Quantitative analysis of the nuclear doxorubicin fluorescence signal was determined by Image J software with or without NH₄Cl treatment. (e) Doxorubicin-loaded MSNPs, fitted with a pH nanovalves, inhibited KB-31 viability efficiently as determined by a MTS assay. Doxorubicin-induced cytotoxicity was partially inhibited by NH₄Cl treatment.