A nanovalve delivery system was designed to meet a specific criterion; i.e., the nanovalve must be closed tightly at pH 7.4—the pH of blood—but self-open in acidifying endosomal compartments, such as lysosomes. In order to achieve this objective, it is necessary to construct stalks that can be attached covalently to the nanopore openings and, in turn, will bind a capping agent, capable of blocking the nanopore openings reversibly. The interaction, a function of the binding constant, must be pH-dependent and change from a large value at pH 7.4 to a small one in response to mild endosomal acidification conditions that will trigger the release of the cap. After experimentation with a number of pH-responsive nanovalve components, a series of aromatic amines were chosen as the stalk and β-cyclodextrin (β-CD) as the cap. The β-CD ring encircles the stalks () as a result of noncovalent bonding interactions under neutral pH conditions and effectively blocks the nanopore openings and traps the included cargo molecules. Lowering of the pH leads to protonation of the aromatic amines, followed by β-CD cap release and cargo diffusion from the nanopores.
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,15 the maximum number of stalks per nanopore (more ...)
Two cell lines were chosen for in vitro biological testing: THP-1 because differentiated THP-1 are macrophage-like and ingest particulate matter in a lysosomal compartment; and KB-31 because it is a doxorubicin-responsive cervical cancer cell that can be used to test effective drug delivery by the mechanized MSNP. In order to achieve the attachment and release of the β-CD caps in the lysosomes of these cells, it was necessary to develop a nanovalve system that responds to the acidification level (pH < 6) that is attainable in the LAMP-1-positive endosomal compartments of THP-1 and KB-31 cells. After a range of aromatic amines that exhibit different pKa values () were tested, the N-methylbenzimidazole (MBI) stalk () was chosen as the best candidate. Upon protonation, the mechanized MSNP release their β-CD caps without causing any cytotoxicity.
Optimization of the Operational pH Conditions of MSNP Fitted with Different Stalks To Define Physiologically Responsive Nanovalvesa
The nanovalves were attached covalently to MSNP, composed of ~100 nm primary nanoparticles with uniform pore size ~2 nm, as indicated by TEM (). The MBI stalks were synthesized and attached covalently to the MSNP surfaces by means of a postsynthesis protocol (). The attachment was confirmed by 13
C-CPMS NMR and 29
Si-CPMS NMR spectroscopies (Supporting Information, Figure S1
). The assynthesized nanoparticles exhibited a hydrodynamic radius of 900 nm in H2
O as a result of aggregation. Optimal dispersion of the nanoparticles was achieved by sonication in a 1 mg mL−1
solution of BSA in H2
O. This procedure is quite effective for nanoparticle (~200 nm) dispersal and ultimately yields negatively charged nanoparticles (zeta potential −7 to −9 mV), most likely as a result of the absorption of proteins from the medium (Supporting Information, Table S1
). The nanoparticle size remains stable over 24 h in the complete cell culture medium.
In order to verify the functioning of the nanovalve system at appropriate pH conditions, the functionalized MSNP were first of all loaded by soaking in concentrated solutions of Hoechst 33342 dye or the fluorescent chemotherapeutic agent, doxorubicin. The nanoparticles were then capped with β-CD in a concentrated aqueous solution and washed carefully. The release of the trapped fluorescent molecules was tested in H2O at pH 7.0 and monitored by time-resolved fluorescence spectroscopy. The capped and loaded nanoparticles were placed in the corner of a cuvette and the surrounding liquid was stirred gently. A probe beam was used to illuminate the liquid above the nanoparticles and to excite the fluorescent molecules as they escaped the nanoparticles. The nanovalves remain closed at pH 7 with no cargo leakage as shown in , but upon decreasing the pH to 5, the fluorescent guest molecules were released. In order to demonstrate that this process is accompanied by β-CD release, the β-CD caps were labeled with pyrene. These fluorescent caps were monitored by fluorescence spectroscopy so as to be able to follow their release into the supernatant (). The rapid release curve shows that β-CD release precedes the release of the Hoechst dye.
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 (more ...)
Curiously, the doxorubicin release was slow and incomplete compared to that of the Hoechst dye. This observation raises the possibility that the cationic nature of this chemotherapeutic agent (pKa
= 8.2) causes it to interact electrostatically with the negatively charged Si–O−
groups in the MSNP nanopores and thus are held relatively tightly to the nanoparticles. In order to alleviate this problem and obtain more rapid and complete release of doxorubicin, the next step in the design strategy was to modify the MSNP surface to prevent electrostatic interactions (Supporting Information, Figure S2a
). Positively charged ammonium groups were attached to the MSNP surface by a cocondensation method.3a
Optimization of the release was achieved by testing a series of concentrations of the ammonium groups to find the cationic density at which the increase in electrostatic interactions—limiting the loading capacity of doxorubicin—is outweighed by an optimal rate of release. Figure S2b (Supporting Information)
shows the loading yield of MSNP with different surface modifications. The MSNP at an ammonium concentration of 7.5% (w/w) show a moderate doxorubicin loading (1.7%, w/w): they can, however, release doxorubicin rapidly so that the MSNP yield the maximum drug delivery per unit time (Supporting Information, Figure S2c
). Thus, functionalization with 7.5% ammonium was used in all subsequent experiments for doxorubicin delivery. shows the improved pH-dependent release profiles of doxorubicin from the ammonium-modified compared to unmodified MSNP (). The fine-tuning of the MSNP pores to optimize cargo specific drug delivery is another important design feature of our system.
In order to prove that the nanovalve integrated system remains functional under biologically relevant conditions, including the presence of buffered salts and growth supplements, Hoechst and doxorubicin release were tested in THP-1 and KB-31 cell culture media. The respective release profiles in RPMI 1640 (THP-1) and DMEM (KB-31) are similar to those in H2
O, and are shown in Supporting Information, Figure S3a and S3b
. The flat baselines and the typical release curves demonstrate that the nanovalve remains operational under culture media conditions.
In order to determine whether the pH-responsive function is maintained intracellularly, it was necessary to demonstrate that MSNP are taken up and capable of localizing in acidifying endosomal compartments. This objective was accomplished by coincubating the FITC-labeled MSNP with THP-1 and KB-31 cells, followed by conducting confocal microscopy in which the late endosomal and lysosomal compartments were stained with TRITC-labeled anti-LAMP-1 antibody. Imaging of both cell types demonstrated >80% colocalization of the green-labeled nanoparticles with the red-labeled lysosomes (). The lysosomal pH values in these cells were determined by measuring the ratios of fluorescence of FITC-dextran at two excitation wavelengths (495/450 nm) and comparing that to a standard pH curve to calculate lysosomal pH.7
The pH values in the acidifying endosomal compartments of THP-1 and KB-31 cells decreased () to 4.6 ± 0.2 and 5.2 ± 0.1, respectively. Introduction of the lysosomal pH neutralizer,8
Cl, elevated () these values to pH 6.1 ± 0.4 and pH 6.2 ± 0.3 in THP-1 and KB-31 cells, respectively. Thus, NH4
Cl treatment provides a way of confirming the role of the acidifying intracellular microenvironment for cargo release.
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 (more ...)
In order to verify these operational limits, confocal microscopy was used to follow the kinetics of Hoechst dye and doxorubicin release in THP-1 and KB-31 cells, respectively. Hoechst-loaded, FITC-labeled MSNP are efficiently taken up () in THP-1 cells. At the earlier times (1 and 3 h), the Hoechst dye is retained in the nanoparticles that localize in the perinuclear regions as fluorescent blue dots, suggesting that the MSNP delivery system does not leak (). However, this visual image changed after 6 h when the Hoechst dye was released to the nuclei of THP-1 () and KB-31 (not shown). This release was accompanied by the disappearance of the blue dots in the perinuclear region, while the FITC-labeled nanoparticles could still be visualized at this site for up to 36 h. Moreover, NH4
Cl treatment was quite effective at confining the Hoechst dye and FITC-labeled nanoparticles to the perinuclear regions without observable nuclear staining (, right panel). To quantitatively measure Hoechst release to the nucleus, Image J software was used to quantify the nuclear fluorescence intensity (). The results confirmed a statistically significant decrease in dye release to THP-1 nuclei by NH4
Cl treatment. Similar results were observed when the cells were treated by bafilomycin,9
another lysosomal pH-neutralizer that inhibits V-ATPases at lysosomal surface10
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 (more ...)
While studying doxorubicin release in KB-31, these cells also efficiently took up FITC-MSNP to the perinuclear regions within 3 h, whereupon there was observable drug release (red fluorescence) to the nucleus (). This release was followed by progressive nuclear changes that reflect the pharmacological effect of the drug, including the formation of apoptotic bodies by 60 h and nuclear fragmentation by 80 h. Again, this visual image was dramatically changed by NH4
Cl treatment, a situation which led to most of the drug being retained inside the nanoparticles and little or no evidence of nuclear staining and cell death (, right panel). This visual impression was further confirmed by Image J software analysis which showed a statistically significant decrease in nuclear doxorubicin staining in the NH4
Cl-treated cells (). A MTS assay was also performed to confirm that NH4
Cl treatment does indeed have the ability to interfere in cytotoxicity (). While the MSNP nanovalve-functionalized MSNP were devoid of cytotoxicity at nanoparticle doses as high as 250 µg/mL, the doxorubicin-loaded nanoparticles resulted in apoptosis that can be confirmed by TUNEL staining11
(Supporting Information, Figure S4
Cl treatment interfered in the induction of programmed cell death (Figure S4, Supporting Information
). Nondrug loaded MSNP, fitted with nanovalves, do not exert detectable cytotoxicity in either KB-31 cells or the 3T3 normal fibroblast cell line (Figure S5, Supporting Information
). This observation is in accordance with the low toxicity of the MBI compound used to construct the nanovalve.12