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Nuclear-targeted therapy has received increasing attention as a potential strategy to improve the therapeutic efficacy of treating cancer. The main agents to the cancer cell nucleus. Nanoparticles as nanocarriers have started to address some of these issues. However, a lack of understanding in how nanoconstructs interact with the nucleus has precluded detailed studies. In this article, we highlight a nanoconstruct composed of gold (Au) nanostars loaded with nucleolin-specific aptamers. This nanoconstruct induced major changes in the nuclear phenotype through nuclear envelope (NE) invaginations. Femtosecond, light-triggered release of the aptamers from the surface of the Au nanostars further increased the number of NE deformations. Cancer cells with more NE folding showed increased apoptosis as well as decreased cell viability. The author’s of this article have revealed that correlation between drug-induced changes in nuclear phenotypes and increased therapeutic efficacy can provide new insight into nuclear-targeted cancer therapy.
Traditional cancer treatments, including chemotherapy, often cause severe side effects in patients [1,2]. Targeted therapy (where tumor cells are targeted via biomarkers overexpressed on the cell surface) has been shown to reduce such adverse effects . Monoclonal antibodies (mAbs) are currently the most common chemotherapeutic agents that bind with high affinity to these cancer markers . Although there are challenges of poor intratumoral mAb uptake and release of drugs from mAb carriers , surface-receptor recognition of ligands (e.g., epidermal growth factors and insulin-like growth factors) have been effectively used ed on their to deliver drugs across the plasma membranes of cancer cells [5,6]. As a result of the success in these studies, recent work has focused on various strategies to improve the efficacy of targeted therapy. Trafficking drug molecules to specific organelles within cancer cells is one of the most recent and promising approaches .
The two most important organelles in drug delivery are the mitochondria and the nucleus. Mitochondria are the powerhouses of cells and key regulators of apoptosis and cell death. Targeting the mitochondria can result in the shutdown of cellular metabolic activities . The main issue in delivering agents to the mitochondria, however, is their highly impermeable inner membranes [7–9]. The nucleus, which possesses genetic material and controls the major biological activities of the cell [10,11], unlike the mitochondria, has a membrane surrounding the nucleus – the nuclear envelope (NE), which allows transport of biomolecules via nuclear pore complexes. Small drug molecules can, therefore, enter the nucleus and potentially cause DNA damage and cell cycle arrest . Although organelle-targeted therapy has the potential to improve targeted therapy in general, two major barriers must be overcome:
Recently, nanoparticles (NPs) of different materials with sizes ranging from 2 to 500 nm have been used as drug-delivery vehicles . As NPs have high surface-to-volume ratios and can be chemically and/or physically outer-surface and inner-core structures, high densities of targeting molecules or anticancer agents can be immobilized on and within NPs . In particular, ligands pack tightly on NP surfaces and, are thus, stabilized. They are, therefore, less likely to degrade in biological environments . In addition, high dosage concentrations in cancer cells can be delivered .
The advantages of NPs in cell-surface targeted therapy can be extended to nuclear-targeted therapy. However, an additional requirement is that either the drug-loaded NPs must be smaller than the size of the nuclear pore complexes (≤50 nm) or that the drugs be released near the nucleus. Small metal NPs (<30 nm) functionalized with multiple nuclear-targeting ligands have resulted in either cell cycle arrest, which induces early apoptosis [15,16], or cell death by unloading the chemotherapeutic agents inside the nucleus [17,18]. In addition, large polymeric NPs (>200 nm) have released drugs into the nucleus by swelling at low pH levels (~2–5) [19–22]. Although these nanocarriers have shown progress in nuclear-targeted drug delivery, there are drawbacks, including:
In this article, we highlight a new two-component nanoconstruct based on aptamer-loaded Au nanostars (Apt–AuNSs; Figure 1A & iB; part [i]) that can solve these problems and open new possibilities for future development of nuclear-targeted therapy.
We believe that the Apt–AuNS is a NP-based system that can serve as a general strategy for the delivery of various drug molecules. By combining the advantages of the properties of an aptamer drug with those of the AuNS nanocarrier, we have designed a drug-stabilized nanoconstruct with potent anticancer activity. For our proof-of-concept studies, we selected the therapeutic aptamer AS1411, since it is currently in clinical trials for the treatment of leukemia and renal cancers [23,24]. This aptamer targets cancer cells by binding with high affinity (Kd is in the picomolar to low nanomolar range) to nucleolin, a nucleolar protein that is also over-expressed on the surfaces of rapidly dividing cells [25,26]. The trafficking ability of nucleolin has been implicated in transporting anticancer agents from the cell surface to the nucleus . By blocking several functions of nucleolin, AS1411 can result in the arrest of DNA repair in the nucleus, as well as destabilization of the anti-apoptotic gene BCL-2, which causes early apoptosis and cell death [26,24]. AuNS particles have unique chemical and physical characteristics that enable them to function as both drug carriers and imaging markers . For example, thiolated oligonucleotides and other small molecules can be densely loaded on the surface of Au NPs ; for AuNSs, we can load approximately 1000 aptamer strands per 30 nm particle. The negative charge of the aptamer creates sufficient electrostatic repulsion to prevent NP aggregation under the high salt concentrations (140 mM) in physiological conditions.
In vitro, fluorescence microscopy is typically used to determine whether nanoconstructs have been targeted and internalized within cancer cells [15,22]. The diffraction-limited resolution of light microscopes, however, is inadequate to observe how drug-loaded NPs interact with cells at the nanoscale, which is critical for understanding how such interactions may correlate to biological changes. Since AuNSs are composed of a high-electron density material, transmission electron microscopy can be used to visualize the relationship between nanoconstructs with cancer-cell nuclei at different time points. After binding to surface nucleolin, Apt AuNS are trafficked through the cytoplasm to the nucleus via cytoplasmic nucleolin (Figure 1B; part [ii]). Although the nanoconstructs were isolated on the cell surface, transmission electron microscopy images indicated that they often clustered within some type of vesicle in the cytoplasm. Interestingly, the formation of folds in the NE occurred where the constructs were within 1 μm of the nucleus. These long folds intruded into the nucleoplasm and resulted in severe deformation in nuclear phenotype. Most interestingly, the NE invaginations were strongly correlated with biological effects. For example, HeLa cells with these nuclear deformations showed a 30% increase in cell death compared with noncancerous MCF-10A cells, which showed no nuclear folds and only minimal cell death .
We discovered that ultra-fast, laser-triggered release of aptamers from AuNSs could exacerbate the severity of NE folding. Femtosecond-pulsed excitation within the biologically transparent window (600–850 nm) and at the surface plasmon resonance of AuNSs can be used to detach aptamers from the AuNS surface . Only short irradiation times (2 s) were required and these short times resulted in very little heating of the surrounding environment  compared with strategies using continuous-wave lasers with irradiation times of 5–10 min for heating [30,31]. Strikingly, the release of aptamers produced nearly double the number of NE folds, compared with controls without laser exposure (and no drug release). This increase in nuclear deformation suggests that released aptamers could have disrupted nuclear function; indeed, immunostaining assays revealed multiple foci of dsDNA breaks in the cell nucleus (Figure 1B; part [iii]), which is likely, since the released aptamer was delivered into the nucleus via the nucleolin shuttle. Biological responses of the cells after the release of aptamer indicated elevations in both apoptosis signals and cancer cell death.
A side-by-side comparison of therapeutic efficacy of drug-loaded AuNSs and free aptamer showed that NPs provide powerful routes for increasing anticancer activity. We found that a twofold increase in apoptosis signals and 80% cell death from the nanoconstruct treatment (0.3 nM) was superior to that of an equivalent concentration of free aptamer (450 nM). Because of the high local concentration of aptamer on each AuNS (1000 strands), more aptamers can make their way to the nucleus via nucleolin without degradation, unlike the free aptamer. Significantly, ultra-fast, light-triggered release of aptamers from AuNSs near the nucleus produced approximately the same apoptosis and viability results as concentrations of free aptamer (10 μM) used in vitro, clinical dosages. Therefore, AuNSs are excellent platforms for delivering localized concentrations of aptamer or other small-molecule drugs that can maximize anticancer efficacy.
Although targeted therapy using NPs as delivery agents is proving increasingly effective, very little research has considered how these nanoconstructs interact with cells in general and the nucleus in particular. Our Apt–AuNS system opens new opportunities for nuclear-targeted therapy in several areas, including:
We expect that new platforms such as Apt–AuNS will provide important insights for the future design of drug-loaded NPs with higher therapeutic efficacy.
Financial & competing interests disclosure
This research was supported by a NIH Director’s Pioneer Award (DP1OD003899) (D.H.M.D., T.W.O.) and the Center of Cancer Nanotechnology Excellence (CCNE) initiative of the NIH under Award Number U54 CA151880 (P.N.S., T.W.O.). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.