Programmed nanoparticle aggregation using molecular beacons.

Nucleic acids can be used as robust building blocks or scaffolds for new nanoarchitectures. The potential of nucleic acids has been demonstrated with the DNA-based construction of a variety of shapes including, for example, an incredible range of two-dimensional patterns, three-dimensional crystals, and even containers with lids that can be opened. DNA scaffolds can be additionally functionalized with proteins to control enzyme cascades. Nucleic acids play a prominent role in integrated nanoparticle–biomolecule hybrid systems and can induce the assembly of other nanoparticles, such as colloidal gold, into large macroscopic and even crystalline materials. Other studies have demonstrated that gold nanoparticles themselves can induce the aggregation of proteins in solution. Alternatively, surfaceenhanced Raman scattering active nanoparticles can be used to detect protein aggregation. Herein we describe a different type of aggregation phenomenon that is based on lipid nanoparticles, rather than metallic ones, and intraparticle forces generated by molecular beacons (MBs), rather than linker-based hybridization of nanoparticle networks. Irreversible aggregation of nanoparticles resulted from the opening of MBs inserted into nanoparticles. We synthesized a MB functionalized with pyropheophorbide (Pyro) along with zero, one, two, or three BlackBerry quencher (BBQ) moieties as previously described. The MB comprised a six-base stem and a 19-base loop (Figure 1a). The 5’ stem of the MB was also complementary to the target sequence since such shared-stem MBs have favorable thermodynamic profiles. Both Pyro and BBQ are hydrophobic, and each additional quenching moiety additionally enhanced the MB hydrophobicity. We hypothesized that these increasingly hydrophobic MBs might insert into lipid nanoparticles such as low-density lipoprotein (LDL). As endogenous nanocarriers, lipoprotein nanoparticles are promising platforms for the delivery of contrast agents and drugs because of their small size, biocompatibility, and capacity to carry a range of cargo and even other small nanoparticles. The LDL nanoparticle concentration was determined by examining the ApoB protein content, as each LDL is stabilized by only one ApoB protein. As shown in Figure 1b, upon incubation of the hydrophobically modified MBs with purified human LDL, hybrid nucleo-lipoprotein nanoparticles were generated and could be assessed using a gel-shift assay. Schematic representations of MB insertion into LDL are shown in Figure 1c. After the negatively charged beacons were inserted into the LDL, the electrophoretic mobility changed. When the beacon lacking any quenchers (0Q MB) was incubated with increasing amounts of LDL, it did not insert effectively into the nanoparticle (Figure 1b). A similar pattern was observed for the single-quencher MB (1Q MB) although at the 7.5:1 beacon/nanoparticle incubation ratio, approximately half the total amount of beacons inserted stably into the nanoparticles. When the 2Q MB was used, the majority of the beacon inserted into the LDL at the 15:1 beacon/nanoparticle Figure 1. MB insertion into nanoparticles. a) Structure of MB modified with Pyro (red) and multiple BlackBerry quencher (BBQ) units (blue). b) Gel-shift assay demonstrating that multiple BBQ units enhance MB insertion into nanoparticles. 15 pmol of MB with the indicated number of BBQ units was incubated with increasing amounts of LDL, and then subjected to agarose gel electrophoresis. Asterisks indicate the migration of the unbound beacon while shifted bands correspond to nanoparticles containing the inserted beacon. c) Schematic illustration of the four different types of MBs with Pyro (P) and BBQ (Q) inserted into the LDL nanoparticles. d) Transmission electron micrographs of negative-stained LDL and LDL with the three-quencher beacon inserted.