Although a variety of small molecule NO donors have been reported effective against different tumor types including pancreatic, colon, and ovarian cancers,24,25,35,36
they have also suffered from highly toxic side effects of the drug byproduct and poor cellular permeability and retention. The development of novel delivery systems for NO donors that may overcome these limitations is of crucial importance to advance the feasibility of NO-based therapies. In this study, we employed NO-releasing silica scaffolds to assess the usefulness of nanoparticle-based NO delivery against ovarian cancer. When compared to control nanoparticles, the NO donor-modified nanoparticles demonstrated enhanced growth inhibition of ovarian tumor cells and preliminary size dependency support the anti-tumor efficacy of NO delivered from silica nanoparticles against ovarian cancer cells as well as the feasibility of materials based from these observations as a novel approach for cancer treatment.
When compared to the small molecule NO donor PYRRO/NO, we found greater anti-tumor activity with the N
-diazeniumdiolated-modified NO-releasing silica nanoparticles. While other small molecule NO donors exist, PYRRO/NO was chosen for study due to its NO release mechanism allowing for a direct comparison between the NO-releasing silica nanoparticles and a previously reported small molecule NO donor. Without changing the structure of the NO donor functionality, we were able to prolong the NO using the silica nanoparticle. Although NO release may be further extended using more recent diazeniumdiolate chemistries (e.g., O2
-arylated diazeniumdiolate), our goal was to keep this chemistry constant. (N
-diazeniumdiolates are characterized by a one-step proton-initiated dissociation mechanism, while O2
-arylated diazeniumdiolates follow a two-step dissociation mechanism (i.e., S
-glutathionylation and protonation).24
The dose of NO required to inhibit tumor cell growth was significantly decreased for the nanoparticles relative to NO derived from the small molecule. Due to an extremely short half-life (~3 s), the effective PYRRO/NO concentration is inherently larger because most of the NO is released prior to the small molecule being taken up by the target cell.29
Since the NO release from the nanocomposite scaffolds is prolonged (t1/2
= 7 min), more NO is available for therapeutic use upon interaction between the nanoparticle and tumor cell. Presence of intracellular NO was confirmed using DAF-2 DA, a widely used NO-sensitive fluorescent probe.37,38
Upon treatment with NO-releasing silica particles and DAF-2 DA, we observed intracellular DAF-2 fluorescence, confirming that our silica particles are capable of delivering significant levels of NO within the cell. Due to the kinetics of NO release, it is likely that some of the payload is released prior to cellular uptake of the particles. However, the presence of DAF-2 fluorescence proves that the nanoparticles are successful in delivering NO to the cell.
Based on our results, NO-releasing silica nanoparticles containing MAP3 are preferable over those composed of AHAP3. Control MAP3 nanoparticles (i.e., non-diazeniumdiolated) demonstrated little to no cytotoxicity in our ovarian cell line panel, while control AHAP3 nanoparticles were slightly cytotoxic. The increased cytotoxicity of AHAP3 over MAP3 nanoparticles is attributed to the presence of the primary amine.39,40
Furthermore, greater aminosilane concentrations were achievable for MAP3 particle systems, thus allowing higher NO payloads. The primary amine of AHAP3 interrupts particle formation at higher concentrations due to hydrogen bonding interactions. These results validate the MAP3 NO-releasing silica nanoparticles as a non-toxic carrier system for large NO payloads.
Nanoparticle size is an important factor in determining the efficacy of a specific anti-cancer therapeutic approach. In the case of drug carriers, size may influence delivery volume, release characteristics, and accumulation site in the body such as tumor, liver, or bone marrow. Previous studies have shown size-dependent tumor-specific delivery of nanoparticles to the tumor microvasculator through the enhanced permeation and retention (EPR) effect.41,42
We found that increasing the size of the nanoparticle from d
= 90 ± 10 nm to d
= 350 ± 50 nm resulted in enhanced preferential cytotoxicity for tumor versus nontumor ovarian cells. Although our initial studies were done in vitro
, with evaluation of only two normal immortalized cell lines and their transformed isogenic counterparts, we believe that these observations are quite significant. Future work will involve synthesizing a range of sizes and investigating not only efficiency of NO delivery but also how particle size affects uptake and distribution within cells.
Many groups have determined that the mechanism of internalization and localization of nanoparticles was influenced greatly by size, surface properties, and cell line-dependence differences.34,43-45
Silica nanoparticles are known to be taken up via endocytosis and localize to endosomes and lysosomes. Thus, as expected, our analyses found that the NO-releasing silica nanoparticles entered the cytosol of the treated cells and localized to late endosomes and lysosomes. Our results show clearly that larger NO-releasing nanoparticles demonstrated enhanced destabilization of mitochondrial function and caspase-induced apoptosis in tumor versus non-tumor cells. The role of NO in the mitochondrial-mediation of apoptosis has been established.46
The nitro-aspirin drug, NCX-4016, induced apoptosis by activation of caspase-3 and cleavage of the substrate PARP in ovarian cancer cells.47
Similarly, the large NO-releasing nanoparticles induced cell death in T80 and more so in T80 H-Ras cells as shown by caspase-3 and PARP cleavage and staining positive for annexin V/PI. The smaller diameter particles exhibited greater cytotoxicity toward T80 cells than the larger particles. Indeed, it has been shown that toxicity of amorphous silica is inversely related to particle size with diameters less than 100 nm showing an increase in cytotoxicity.48,49
This phenomenon is attributed to the fact that the surface area to volume ratio increases exponentially as particle size decreases. More atoms or molecules are expressed on the surface of smaller diameter particles per volume compared to larger particles therefore increasing the biological activity of the smaller nanoparticle. The enhanced cytotoxicity and caspase induced-apoptosis seen in T80 H-Ras cells after treatment with the large NO-releasing nanoparticles may be attributed to the greater concentration of NO released by these scaffolds and/or NO targeting Ras-driven apoptotic pathways.
While we have shown that NO-releasing silica nanoparticles are effective against ovarian tumor cells, modifications must be made to enhance the therapeutic potential due to the short half-life of NO release. Addition of an outer shell (i.e. TEOS) could temporarily protect the diazeniumdiolate functionalities and delay NO release. Alternatively, encapsulation of the NO-releasing silica particles within a liposome would suspend NO release until obliteration of the liposome. Future studies will need to determine whether size selectivity is maintained in the much more complex in vivo
environment, where pharmacodynamic and phamacokinetic parameters will also influence nanoparticle-delivered NO anti-tumor activity and selectivity. Furthermore, prior to in vivo
testing, the surface of the particles will be modified with polyethylene glycol (PEG) to increase biocompatibility and blood circulation times.50,51