Multiplexed mRNA Sensing and Combinatorial-Targeted Drug Delivery Using DNA-Gold Nanoparticle Dimers

The design of nanoparticulate systems which can perform multiple synergistic functions in cells with high specificity and selectivity is of great importance in applications. Here we combine recent advances in DNA-gold nanoparticle self-assembly and sensing to develop gold nanoparticle dimers that are able to perform multiplexed synergistic functions within a cellular environment. These dimers can sense two mRNA targets and simultaneously or independently deliver one or two DNA-intercalating anticancer drugs (doxorubicin and mitoxantrone) in live cells. Our study focuses on the design of sophisticated nanoparticle assemblies with multiple and synergistic functions that have the potential to advance sensing and drug delivery in cells.

are superior ligands because of their biological relevance, high specificity, selectivity and versatility in conjunction with the ease of their chemical manipulation. 5 Several groups have used oligonucleotide functionalized AuNPs to create nanoparticle dimers, trimers and tetramers with tunable nanoparticle distances as well as mesoscale structures containing millions of nanoparticles in precise arrangements (e.g. body-centered cubic and face-centered cubic). [9][10][11][12][13] Recently our group developed ligation methods to covalently bind DNA-AuNP assemblies, enhancing their stability under DNA denaturing conditions or biologically complex environments. 14,15 In one of these methods, a copper-free click chemistry strategy was utilized to permanently ligate DNA-AuNP conjugates. Synthetic oligonucleotides modified by an alkyne or azide group were brought into close proximity via a templating splint strand. Partial complementarity to both modified oligonucleotides resulted in successful oligonucleotide hybridization bringing in close proximity the alkyne and azide groups, catalyzing a ligation reaction. This approach to manipulating AuNP assemblies allowed the formation of AuNP dimers and trimers in high yield, which were stable even under DNA denaturing conditions. 15 Beside the developed strategies to accurately organize and manipulate gold nanoparticle assemblies using DNA, there has been significant progress in the design of individual oligonucleotide coated gold nanoparticles for biomedical applications. For example, Mirkin and co-workers synthesized AuNPs densely functionalized with a 3d monolayer of synthetic oligonucleotides termed spherical nucleic acids (SNAs). 8,16 It was demonstrated that SNAs are readily taken up by cells mainly via a caveole mediated endocytosis pathway. [17][18][19][20] A striking observation of their study was that the oligonucleotides attached to the nanoparticle surface did not degrade by endocellular enzymes possibly due to the highly ionic and steric microenvironment around the nanoparticles, which prevented the function of DNase enzymes. 5,21 SNAs were further developed to include short, dye-functionalized oligonucleotide strands that could sense mRNA in cells in real time. [22][23][24][25][26] These particles were used to detect the survivin mRNA transcript. The survivin protein functions to inhibit apoptosis of the cell whilst also regulating cell proliferation and is expressed in high numbers in many human cancers. SKBR3 (human breast cancer) and HeLa (human cervical cancer) cells were treated with nanoparticle probes for the detection of survivin mRNA and a 2.5-fold higher fluorescence signal was detected compared to treatment with non-targeting nanoprobes. 23,27 SNAs have also been employed in cancer stem cells (CSC), a distinct subpopulation within a tumor, which is thought to drive tumor progression. Owen et al. designed a probe specific for the detection and isolation of viable CSC using flow cytometry via the specific detection of Nanog mRNA, a marker that is highly expressed in cancer stem cells and that correlates with patient survival. 28 On the other hand, Hendrix et al. monitored the expression of Nodal mRNA in melanoma, which has been shown to underlie unregulated cell growth, metastasis and the CSC phenotype. 29 Li and co-workers adapted SNAs in order to monitor the expression of Runx2 and Sox9 mRNA, two targets that can be used to assess the osteogenic differentiation of human bone marrow derived mesenchymal stromal cells (hBMSCs). 30 Krane et al. demonstrated the expression of Nanog and GDF3 in embryonic stem cells and induced pluripotents stem (iPS) cells of murine, human and porcine origin. Furthermore, they also monitored the expression of GAPDH, a common house-keeping gene, in somatic cells. 31 In another category of studies, the design of the SNAs was altered to include hairpin forming oligonucleotides containing a terminal dye, which were then attached to a gold nanoparticle quencher to act as fluorescent molecular beacons upon the detection of a specific mRNA. For example, Sun and co-workers designed a hairpin nanoparticle probe targeting exon8 of BRCA1 mRNA, a human tumour suppressor gene that plays an important role in repairing damaged DNA, whilst Gu and co-workers focused on the detection of STAT5B mRNA, which provides insight into tumour progression, in MCF 7 cells (human breast cancer). 24,25 As a first example of a multiplexed nanoprobe, Prigodich et al. designed an SNAs probe capable of detecting, simultaneously, two different mRNA targets related to survivin and actin, by monitoring two separate fluorescent outputs. 32 Tang and co-workers adapted this approach and developed nanoparticle probes for the simultaneous detection of three and four intracellular mRNA biomarkers (c-myc, TK1, Ga1Nac-T and survivin mRNA) all related to the process of tumor progression. 33,34 However, the ratio among the sense oligonucleotide strands on the nanoparticle was not controlled, a limitation that can influence significantly the efficiency of nanoparticles to detect all the relevant mRNA targets.
Recently, our group reported how SNAs could be designed and finely tuned to achieve localized and specific endocellular drug delivery upon the detection of a specific mRNA target. 35 Using those DNA-coated gold nanoparticles we were able to selectively kill only mesenchymal cells without affecting epithelial cells demonstrating that they can deliver their cargo only to cells expressing a specific mRNA signature. 36 In this work, we combine advances in nanoparticle self-assembly and nanoparticle functionalization to develop multiplexed DNA-gold nanoparticle dimers that are able to enter cells and coordinate the delivery of two different DNA intercalating drugs into the local cellular microenvironment with high selectivity and specificity. The coordinated drug delivery is feasible by qualitatively detecting independent mRNA signatures. Superiority in this design comes from the ability to precisely control the number of oligonucleotides on the surface of each nanoparticle and therefore the number of intercalating drugs that can be loaded onto the probe. This nanoparticulate design is shown to be successful in the combinatorial and highly selective treatment of cells to achieve the highest drug delivery efficiency at a localized area.

RESULTS AND DISCUSSION
Design of DNA-gold nanoparticle dimers. The formation of gold nanoparticle dimers and their covalent linking using copper-free click chemistry is described into detail in supporting information Scheme S1. Briefly, BSPP coated gold nanoparticles modified with one oligonucleotide strand were prepared and purified via agarose gel electrophoresis in order to isolate gold nanoparticles bound to only one oligonucleotide (monoconjugates). Each batch of monoconjugates was chemically modified with either an azide group (Linker strand 1) or an alkyne group (Linker strand 2) (see supporting information Figure S1 for chemical structures).
Then the particles were functionalised with a shell of oligonucleotide sense strands (Sense strands 1 and 2), designed to capture a specific mRNA target (see supporting information Table   S1 and S2 for oligonucleotide sequences). Nanoparticles were then linked together via hybridization of linker strand 1 and linker strand 2, which resulted in spontaneous DNA ligation, and purified by gel electrophoresis under DNA denaturing conditions in order to disregard particles that did not form dimers or that were not chemically ligated. Successful dimer formation was also assessed by Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) (see supporting information Figure S5). For mRNA detection, short fluorophore-modified oligonucleotides (Flare strands 1 and 2) were added to the sample and hybridized to their complementary sense strands (see supporting information Table S2 for oligonucleotide sequences). Scheme 1 shows the design of the fully assembled gold nanoparticle dimers and illustrates the process of mRNA detection. The nanoparticle dimer design includes sense strands (attached to the AuNP surface), bearing a 5' fluorophore modification, which are partially hybridised to shorter oligonucleotide complements, termed flare strands, chemically modified at the 3' end with another dye. Once hybridised, the fluorescence of all fluorophores is quenched, due to the close proximity to the AuNP surface. 37,38 However, once the target mRNA binds to the corresponding sense sequence via competitive hybridization, the concomitant displacement of the flare can be detected as an increase in fluorescence at the specific wavelength of the fluorophore. 22,23,27,32,39 . The fluorophore on the sense strand in this design acts as a reporter that ensures the integrity and specificity of the system. A FAM signal should not be detectable unless DNA degradation due to the presence of nucleases has taken place. Scheme 1. Schematic illustration of the multiplexed nanoparticle dimer and the process of mRNA detection. When the target mRNAs bind to the sense strand a short oligonucleotide strand is released resulting in an increase in the fluorescence signal, which was previously quenched by the AuNP core. Flare strand 1 is modified with Cy3 (Dye 1) and flare strand 2 is modified with a Cy5 dye (Dye 2) whereas both sense strands have been modified with FAM ( Prior to testing the function of nanoparticle dimers within a cellular environment, their stability against common nucleases such as cytosolic DNase I and lysosomal DNase II was investigated.
Nanoparticle dimers were incubated with DNase I and DNase II and the integrity of the oligonucleotide shell was determined by monitoring the fluorescence output from the fluorophore-modified sense strands. The stability of the nanoparticles dimers against DNA enzymatic degradation was also monitored via gel electrophoresis against single nanoparticle probes. Figure S3 shows that in both cases our nanoparticle dimers not only retained their dense oligonucleotide shell but also their dimeric structure. This is most likely due to the high density of the oligonucleotides on the AuNP surface, creating steric effects as well as high local ionic strength around nanoparticles preventing enzymatic degradation. 5 In order to demonstrate the ability of the nanoparticle dimer to sense more than one target mRNA simultaneously, we employed the adenocarcinoma-derived human alveolar cell line A

This cell line expresses both keratin 8 and vimentin as observed from RT-qPCR and
immunostaining experiments (see supporting information Figure S9 and S10). Therefore both of these mRNAs are expected to be detected in live A 549 cells. Figure 1C shows that after incubation of the nanoparticle dimers with A 549 cells, two fluorescence signals (red and green) corresponding to the detection of both keratin 8 and vimentin mRNAs were observed. To further confirm the specificity of the particles for targeted drug release, all three cell lines were incubated with "non-targeting" scramble oligonucleotide functionalized nanoparticle dimers.
Sense strands on these particles (for oligonucleotide sequences, see Table S2 at the supporting information) were not designed to target any cellular mRNA and therefore no fluorescence signal should be observed corresponding to flare release. Figure 1D-F shows that in all three cases, no fluorescence signal was detected from either the flare strand or the anchored sense strand. Although it has been shown that several types of functional nanoparticles, mainly coated by peptides or other transfection agents, can escape endosomes during cellular endocytosis via various ways, a mechanism for the escape of SNAs from endosomes has not been reported yet. 18,40,41 Nevertheless, experiments previously reported by our group and others indicated that SNAs are highly specific for the detection of endocellular mRNAs as well as in the release of DNAintercalating drugs. 23,[33][34][35] In order to identify the location of the nanoparticle dimers within a cell and to confirm that the DNA between the nanoparticles does not degrade; we analyzed several ultra-thin sections of cells incubated with the nanoparticle dimers and imaged via TEM. Our TEM analysis shows that about 1-3 % of nanoparticle dimers are found outside endosomes (see supporting information Figure S13), concluding that these ones are responsible for the highly specific signals we obtained. The two drugs were incorporated into the nanoparticle dimers following a two-step process.
First, DOX was mixed together with the flare strand 1 as well as the dimer nanoparticles and a heat-cool step was performed to smoothly intercalate the DOX between the sense and flare strand 1 duplex. The sample was purified from excess of free drug, after which the same nanoparticle dimers were mixed together with MXT and the flare strand 2. To avoid the release of DOX from the nanoparticle dimers, while at the same time maximizing the hybridization of sense and flare strands 2 and therefore the intercalation of MTX, the ionic strength of the solution was increased by adding MgCl 2 (see supporting information Table S4). This increase in the ionic strength allowed an efficient intercalation of MXT between the sense/flare duplex that detects the vimentin mRNA. Successful drug loading was first evaluated in vitro. After purification, the nanoparticle dimers loaded with the drugs were heated to 80 ºC for 10 minutes.
An increase in temperature resulted in dehybridisation of the flare strands and release of the intercalated drugs, which was monitored through their specific fluorescent signatures. Under the experimental conditions used here, it was determined that the average DOX loading was 34 ± 4 and MTX loading was 30 ± 1 molecules per nanoparticle (see supporting information -section (S-VIII)).
Having observed that the nanoparticle dimers are highly specific to mRNA detection (see figure   1), and can be uploaded with two drugs simultaneously, we used them for drug delivery in live   Table S5). The cell viability of all three cell-lines after an 18 h incubation period was evaluated using trypan blue as shown in Figure 3. A range of conditions was tested, including nanoparticle dimers with and without drugs, "non-tageting" scramble nanoparticle dimers loaded with drugs and free DOX and MXT without nanoprobes.
While the nanoparticle dimers without drugs did not exhibit any toxicity, once loaded with the drugs a dramatic reduction in viable cells was observed for probes bearing specific mRNA sense strands. 16HBE cell lines showed a decrease in viability of over 80 % when incubated with the nanoparticle dimers while the viability of the MRC5 cells' was reduced by up to 92 % ( Figure   3A and B). On the other hand, in A 549 cancerous cells the decrease in cell viability was at 70 %. Although one would expect that A 549 cells would be less viable because of the release of two drugs in cells at the same time, this was not observed in our experiments. This observation may be supported by recent literature which states that lung cancer cells, such as A 549 cells, overexpress cytoprotective transcription factors thus exhibiting an increased resistance to anticancer drugs. 44 Moreover, in order to prove the specificity of the nanoparticle dimers, all three cell-lines were also incubated with the same "non-targeting" scrambled nanoparticle dimers loaded with both DOX or MXT and no significant decrease in cell viability was observed. When using high concentrations of free DOX or MXT that had not been loaded onto a nanoparticle carrier, a reduction of viability of around 20 % was observed, much less than when administering these drugs within the nanoparticle dimers. These observations emphasize the specific and efficient delivery of DOX and MTX when using the gold nanoparticle dimer platform.

Supporting information
The Supporting Information is available free of charge on the ACS publication website at DOI: