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Multiplexed mRNA Sensing and Combinatorial-Targeted Drug Delivery Using DNA-Gold Nanoparticle DimersClick to copy article linkArticle link copied!
- Maria-Eleni KyriaziMaria-Eleni KyriaziPhysics and Astronomy, Faculty of Physical Sciences and Engineering, University of Southampton, Southampton SO171BJ, United KingdomMore by Maria-Eleni Kyriazi
- Davide GiustDavide GiustPhysics and Astronomy, Faculty of Physical Sciences and Engineering, University of Southampton, Southampton SO171BJ, United KingdomMore by Davide Giust
- Afaf H. El-SagheerAfaf H. El-SagheerDepartment of Chemistry, Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, United KingdomChemistry Branch, Department of Science and Mathematics, Faculty of Petroleum and Mining Engineering, Suez University, Suez 43721, EgyptMore by Afaf H. El-Sagheer
- Peter M. LackiePeter M. LackieClinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton SO16 6YD, United KingdomMore by Peter M. Lackie
- Otto L. MuskensOtto L. MuskensPhysics and Astronomy, Faculty of Physical Sciences and Engineering and Institute for Life Sciences, University of Southampton, Southampton SO171BJ, United KingdomMore by Otto L. Muskens
- Tom BrownTom BrownDepartment of Chemistry, Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, United KingdomMore by Tom Brown
- Antonios G. Kanaras*Antonios G. Kanaras*E-mail: [email protected]Physics and Astronomy, Faculty of Physical Sciences and Engineering and Institute for Life Sciences, University of Southampton, Southampton SO171BJ, United KingdomMore by Antonios G. Kanaras
Abstract
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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.
Copyright © 2018 American Chemical Society
Progress in the field of nanoscience has rendered available a variety of inorganic nanoparticles in terms of their chemical composition and morphology and therefore their properties. Moreover, scientists have gained a better understanding of the surface chemistry of nanoparticles, which allowed the development of protocols to produce colloidally robust and functional nanoparticulate systems, an essential step toward effective applications. (1−4) Among various types of nanoparticles, gold nanoparticles (AuNPs) are broadly utilized in research and applications because of their intrinsic optical properties and the ease of surface chemical modification with various types of ligands. (5) In recent years, advances in experimental protocols to functionalize AuNPs with thiol-modified oligonucleotides have enabled the design of sophisticated nanoparticles as well as the formation of precisely organized nanostructures. (6−8) Oligonucleotides 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). (9−13) 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 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 AuNP assemblies using DNA, there has been significant progress in the design of individual oligonucleotide-coated AuNPs 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. (17−20) 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. (22−26)
These particles were used to detect the survivin mRNA transcript. The survivin protein functions to inhibit apoptosis of the cell while 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 AuNP 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 tumor suppressor gene that plays an important role in repairing damaged DNA, while Gu and co-workers focused on the detection of stat5b mRNA, which provides insight into tumor 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 AuNPs, 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-AuNP 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
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Design of DNA-AuNP Dimers
The formation of AuNP dimers and their covalent linking using copper-free click chemistry is described into detail in Supporting Information Scheme S1. Briefly, BSPP-coated AuNPs modified with one oligonucleotide strand were prepared and purified via agarose gel electrophoresis in order to isolate AuNPs 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 functionalized 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 strands 1 and 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 AuNP 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 hybridized to shorter oligonucleotide complements, termed flare strands, chemically modified at the 3′ end with another dye. Once hybridized, 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
Scheme 1. Schematic Illustration of the Multiplexed Nanoparticle Dimer and the Process of mRNA Detectiona
aWhen 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 (Dye 3).
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. Supporting Information 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)
Multiplexed mRNA Sensing
In our experiments, the oligonucleotide sequences on the surface of the nanoparticle dimers were designed to detect keratin 8 and vimentin mRNAs. The 16HBE cell line was used as a model for epithelial cells that strongly express keratin 8 while not expressing vimentin.
Figure 1
Figure 1. Confocal microscopy images of cells incubated with (A–C) nanoparticle dimers and (D–F) “non-targeting” scramble nanoparticle dimers. Sixteen HBE cells express only keratin 8, and MRC 5 cells express only vimentin. A549 expresses both keratin 8 and vimentin. A fluorescence signal corresponding to the presence of: keratin 8 mRNA (A), vimentin mRNA (B), and both vimentin and keratin 8 mRNA (C) is observed. All three cell lines display no response when incubated with nanoparticle dimers designed with “non-targeting” scramble sequences. Color guide: Keratin 8 “flare” strand, green; vimentin flare strand, red; vimentin and keratin 8 sense strand, blue; scrambled flare strand, green; scrambled sense strand, blue; nuclear counterstain, white. Scale bars are 15 μm.
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 A549. 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 A549 cells. Figure 1C shows that after incubation of the nanoparticle dimers with A549 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 DNA-intercalating drugs. (23,33−35) 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 ultrathin 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.
Targeted Drug Delivery
After successfully testing the ability of the nanoparticle dimers to sense two mRNA signatures independently or simultaneously, we assessed their ability to act as a drug delivery vehicle of more than one drug. Doxorubicin (DOX) and mitoxantrone (MXT) were chosen as widely recognized anticancer drugs. Both bind within a DNA duplex by intercalation of the tetracyclic region (DOX) and anthraquinone ring (MXT) into the DNA base pairs. (42,43)
Scheme 2 shows the design of the nanoparticle dimers as drug delivery vehicles for DOX and MXT. DOX was intercalated in the DNA duplex that detects the keratin 8 mRNA, whereas MXT was intercalated in the DNA duplex that detects vimentin mRNA. In order to ensure that the detection of any fluorescent signal was solely due to the release of the drugs, no additional fluorophore labels were used in this experiment apart from DOX and MTX, which by themselves have specific fluorescent signatures. When intercalated in the DNA duplex, close to the AuNP surface, their fluorescence is quenched; however, when a target mRNA binds to the correspondent sense sequence, the flare is displaced leading to the release of the intercalated drug, which is detected as a corresponding increase in the fluorescence signal of the drug via confocal microscopy.
Scheme 2
Scheme 2. Schematic Illustration of Nanoparticle Dimer Used in Drug Release Experimentsa
a(Left) Dimer loaded with DOX (Drug 1) and MTX (Drug 2). No fluorophores were used in flare and sense strands. Both DOX and MTX have fluorescent properties, which are quenched due to the close proximity to the AuNP core. When the target mRNA binds to the sense strand, the intercalated drugs are released (right) causing an increase in the fluorescence signal.
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
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 cells. Model epithelial (16HBE), mesenchymal (MRC 5), and cancerous (A549) cell lines were treated with the nanoparticle dimers loaded with both DOX and MXT. By relying on the fluorescence properties of both drugs, the release of DOX and MXT was monitored via confocal microscopy. Figure 2 shows confocal images where DOX and MXT were only released in the presence of the corresponding target mRNA. When the nanoparticle dimers were incubated with 16HBE epithelial cells, only a fluorescence signal corresponding to the release of DOX was observed (Figure 2A). In MRC 5 cells, only the MXT was released due to the detection of the vimentin mRNA, while the nanoparticle responsible for the detection of keratin 8 mRNA retained its payload (Figure 2B). On the other hand, when the nanoparticle dimers were incubated with A549 cells, which express both vimentin and keratin 8, a fluorescence signal corresponding to the release of both drugs was observed (Figure 2C). The specificity of the delivery of both DOX and MXT was further confirmed by using drug loaded “non-targeting” scramble nanoparticle dimers (see Supporting Information Table S2 for sequences). Each cell line was incubated with nanoparticle dimers loaded with both DOX and MXT. The scramble sequences on the nanoparticles do not have a recognition site for the mRNAs in these cells, and therefore the drugs were not released. From the absence of fluorescence in the confocal images (Figure 2D–F) it is evident that the “non-targeting” scramble nanoparticle dimers were able to retain their drug payload.
Figure 2
Figure 2. Confocal microscopy images of live cells incubated with nanoparticle dimers. The nanoparticle dimers were loaded with DOX and MXT (left column). The 16HBE cells show release of only DOX, corresponding to the presence of keratin 8 mRNA (see Figure 1). In MRC 5 cells, MTX was released corresponding to the presence of vimentin mRNA. In A549 cells, both keratin 8 and vimentin mRNA were detected, therefore both drugs MTX and DOX were released. Scramble nanoparticle dimers (D–F) did not display any fluorescence signal indicating that they retained their drug payload. Color guide: DOX release, green; MXT release, red; nuclear counterstain, white. Scale bar is 15 μm.
Cell viability assays were conducted to determine the effect of drug to the cells. For the purpose of this experiment, the loading of DOX and MTX in the nanoparticle dimers was increased. Nanoparticle dimers were hybridized with an excess of flares strands resulting in 118 ± 4 and 116 ± 2 flares hybridized for keratin 8 and vimentin, respectively. This led to an increase in the drug payload of both DOX (117 ± 2) and MXT (113 ± 3) molecules per nanoparticle (see Supporting Information 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.
Figure 3
Figure 3. Trypan blue cell viability assay of (A) 16 HBE (B) MRC 5 and (C) A 549 cells incubated with various nanoparticle designs. Cells were incubated with: (2) nanoparticles sensing all mRNAs (gmRNA); (3) nanoparticles functionalized with scramble “non-targeting” oligonucleotides loaded with DOX and MXT; (4) free DOX; (5) free MTX; (6) nanoparticle dimers designed to sense keratin8 and vimentin mRNA loaded with DOX and MTX. Data are shown as mean ± SEM (n = 3). P-values were determined by a one-way ANOVA. *p < 0.05, ****p < 0.0001.
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 A549 cancerous cells, the decrease in cell viability was at 70%. Although one would expect that A549 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 A549 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 AuNP dimer platform.
Conclusion
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In conclusion, we have successfully shown that dimers of AuNPs can be employed to sense two different mRNA targets within cells and release two different drugs in response to the presence of specific mRNA signatures. Furthermore, we have shown that selective drug release results in the death of cells expressing the specific mRNA target. This design of nanoparticle assemblies that can perform multiplexed synergistic roles of sensing and drug delivery triggered by specific biological fingerprints within cells is of great interest because it has the potential to advance therapeutic treatments by reducing nonspecific targeting and toxicity.
Experimental Methods
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Synthesis of 15 ± 1.5 nm AuNPs
A sodium tetrachloroaurate (1 mL, 100 mM) solution was brought to the boil while stirring. To this, a sodium citrate solution (5 mL, 2%wt) was added, and the solution was stirred for a further 15 min. Then, the reaction mixture was left to cool down while stirring. Once room temperature was reached bis-sulfonatophenylphosphine (BSPP, 42 mg in 2 mL of Milli-Q water) was added to the reaction mixture, which was left stirring overnight. Large aggregates were removed by filtering the solution through a 0.45 Millipore filter, and a concentrated solution of sodium chloride was added to the reaction mixture until a color change from wine red to gray/blue was observed indicating particle precipitation. Particles were purified through two sets of centrifugation at 10,000 rpm and redispersed in 3 mL of Milli-Q water.
Multiplexed Nanoparticle Dimers
BSPP-coated 15 nm AuNPs (1 mL, 50 nM) in attachment buffer (20 mM PBS, 5 mM NaCl) were incubated with thiol-modified single strand DNA (ssDNA) bearing alkyne or azide moieties at a 1:1 molar ratio. Additional BSPP (10 μL, 1 mg/20 μL) was added to the reaction mixture, which was then left to shake gently for 1 h. AuNPs with one DNA strand were purified by gel electrophoresis (2% agarose gel, 10 V/cm, 1 h) and stored in water.
A salt aging method was then employed to achieve a dense shell of “sense” DNA strands (targeting vimentin or keratin 8) around each batch of AuNPs functionalized with one DNA strand (monoconjugates). (8) Briefly, BSPP-coated 15 nm gold DNA monoconjugates (1 mL, 10 nM) in water were incubated with thiol-modified DNA strands (3 μM, 1 mL), and the reaction mixture was left to shake for 24 h. BSPP (10 μL, 1 mg/20 μL), phosphate buffer (0.1 M, pH 7.4), and sodium dodecyl sulfate (SDS, 10%) were then added to the nanoparticles’ solution to achieve final concentrations of 0.01 M phosphate and 1% SDS respectively. A final salt concentration of 0.30 M was achieved by 6 additions of a NaCl (2 M) solution over a period of 8 h. The resulting DNA-coated AuNPs were purified by three steps of centrifugation (16,400 rpm, 20 min) and were stored at 4 °C in a hybridization buffer (5 mM phosphate buffer, 80 mM NaCl).
Nanoparticle dimers were formed by combining the DNA-coated AuNP batches at a molar ratio of 1:1 and by heating at 80 °C for 5 min and then cooling to room temperature. The close proximity between the azide and alkyne moieties after hybridization resulted in the chemical ligation of DNA between the two nanoparticles. The resulting product was subjected to denaturing conditions via incubation with formamide (50% v/v). The solution was heated to 80 °C for 15 min followed by immediate purification by gel electrophoresis (1.75% agarose gel, 10 V/cm, 30 min) and stored in PBS.
For flare hybridization, nanoparticle dimers (3.75 nM, 200 μL) were mixed with an excess of the complementary flare strands (75 nM, 200 μL). The solution was heated up to 55 °C for 5 min, followed by slow cooling to room temperature to allow hybridization to occur. Samples were purified by two rounds of centrifugation (16,400 rpm, 15 min) or until no background fluorescence was observed from flare strands within the solution and redispersed in PBS.
Drug Intercalation
For drug incorporation, nanoparticle dimers in PBS (3.75 nM, 200 μL) were first incubated with an excess of DOX (1 mg/4 mL) and keratin 8 flare strands (75 nM, 200 μL). The solution was heated up to 60 °C for 5 min and left to cool down to room temperature. The probes were purified by three rounds of centrifugation (16,400 rpm, 4 °C, 30 min) or until no fluorescence from DOX was detectable in the supernatant. The dimer nanoparticles were dispersed in a buffer (Tris 10 mM, MgCl2 7.5 mM and NaCl 300 mM) and incubated with an excess of MXT (1 mg/4 mL) and vimentin flare strands (75 nM, 200 μL). For cell viability experiments, nanoparticle dimers were loaded with more DOX and MXT by increasing the number of flare strands attached per nanoparticle for both keratin 8 (225 nM, 200 μL) and vimentin (225 nM, 200 μL). MgCl2 (7.5 mM, 200 μL) and an increased salt concentration (300 mM, 200 μL) were used. The solution was sonicated for 5 min and then left to shake overnight. After 3 rounds of purification by centrifugation (16,400 rpm, 4 °C, 30 min) and ensuring that there is no MXT fluorescence in the supernatant, the nanoparticle dimers were dispersed in PBS and used in further cellular experiments.
Cell Viability Assay
Cells were seeded in 24-well plates up to 90% confluency. Then they were incubated with the fully assembled nanoparticle dimers (500 μL, 32 nM) for 18 h. Nanoparticle dimers were loaded with an excess of keratin 8 and vimentin flare as well as an excess of DOX and MXT according to a previously stated method. Then cells were washed with Hank’s salt (HBSS) followed by incubation with trypsin (250 μL, 0.25% trypsin 0.01% EDTA solution) to detach cells from the surface. Any solution removed from the well prior to trypsinization was kept in an eppendorf and combined with the trypsinized cells, ensuring that cells detached from the surface prior to trypsinization were included in our interpretation of the cell viability. After 5 min incubation at 37 °C, the trypsin was inhibited with cell culture medium. This was combined with the previously removed medium, and the cells were pelleted by centrifugation (5000 rpm, 5 min). Pelleted cells were dispersed in fresh medium (10 μL) along with an equal amount of trypan blue (10 μL). Cells were counted using a Neubauer hemocytometer where cell viability was assessed by counting both live (clear cytoplasm) and dead (blue-colored cytoplasm) cells. Cell viability was calculated based on triplicates of experiments where the number of cells counted per experiment ranged between 624 and 2033.
Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b08620.
Additional experimental detail and data including nanoparticle characterization (TEM, DLS, melting curves, nuclease assays), detailed oligonucleotide sequences and further intracellular studies (confocal images, TEM cross sectioning, immunofluorescent labeling, RT-qPCR); the raw data for this work can be found at http://doi.org/10.5258/SOTON/D0448 (PDF)
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Author Information
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- Antonios G. Kanaras - Physics and Astronomy, Faculty of Physical Sciences and Engineering and Institute for Life Sciences and , University of Southampton, Southampton SO171BJ, United Kingdom;
http://orcid.org/0000-0002-9847-6706;
- Afaf H. El-Sagheer - Department of Chemistry, Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, United Kingdom; Chemistry Branch, Department of Science and Mathematics, Faculty of Petroleum and Mining Engineering, Suez University, Suez 43721, Egypt;http://orcid.org/0000-0001-8706-1292
- Otto L. Muskens - Physics and Astronomy, Faculty of Physical Sciences and Engineering and Institute for Life Sciences and , University of Southampton, Southampton SO171BJ, United Kingdom;http://orcid.org/0000-0003-0693-5504
- Tom Brown - Department of Chemistry, Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, United Kingdom;http://orcid.org/0000-0002-6538-3036
Acknowledgments
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Authors would like to thank the Leverhulme Trust (ref RPG-2015-005) and BBSRC (BB/N021150/1) for funding of this project. ATDBio is gratefully acknowledged for technical support regarding the oligonucleotide synthesis. We thank the staff at the Biomedical Imaging Unit and Primary Ciliary Dyskinesia Research Group, University of Southampton, for their assistance and technical support as well as Dr. Alastair Watson and Dr. C. Mirella Spalluto for providing us with the A549 cell line.
References
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- 1Yang, X.; Yang, M. X.; Pang, B.; Vara, M.; Xia, Y. N. Gold Nanomaterials at Work in Biomedicine. Chem. Rev. 2015, 115, 10410– 10488, DOI: 10.1021/acs.chemrev.5b00193Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtlyjsrzE&md5=42d2194c37216db6f9acdcd3b6bcb91bGold Nanomaterials at Work in BiomedicineYang, Xuan; Yang, Miaoxin; Pang, Bo; Vara, Madeline; Xia, YounanChemical Reviews (Washington, DC, United States) (2015), 115 (19), 10410-10488CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Owing to their inherent bioinertness and tunable optical properties, Au nanomaterials have shown tremendous promise in a wide variety of biomedical applications, including sensing,imaging, diagnostics, drug delivery, gene delivery, and cancer treatment.
- 2Shenashen, M. A.; El-Safty, S. A.; Elshehy, E. A. Synthesis, Morphological Control, and Properties of Silver Nanoparticles in Potential Applications. Part. Part. Syst. Char. 2014, 31, 293– 316, DOI: 10.1002/ppsc.201300181Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXkt1Sju7c%253D&md5=e1a3e3cb95619f5ab0499e1dbd0f5d77Synthesis, Morphological Control, and Properties of Silver Nanoparticles in Potential ApplicationsShenashen, Mohamed A.; El-Safty, Sherif A.; Elshehy, Emad A.Particle & Particle Systems Characterization (2014), 31 (3), 293-316CODEN: PPCHEZ; ISSN:1521-4117. (Wiley-VCH Verlag GmbH & Co. KGaA)Nanostructured materials, esp. nanoparticles (NPs), of noble metal NPs such as silver (Ag) have been the focus of research in recent decades because of their distinct phys., chem., and biol. properties. These materials have attracted considerable attention because of their potential applications, such as catalysis, biosensing, drug delivery, and nanodevice fabrication. Previous studies on Ag NPs have clearly demonstrated that their electromagnetic, optical, and catalytic properties are strongly influenced by their shape, size, and size distribution, which can be varied by using different synthetic methods, reducing agents, and stabilizers. The valuable optical properties of Ag NPs have allowed for new approaches in sensing and imaging applications, offering a wide range of detection modes, such as colorimetric, scattering, and surface-enhanced Raman scattering techniques, at extremely low detection limits. Here, an overview of the various chem., phys., and biol. properties of Ag NP fabrication approaches to obtain the various shapes and sizes is presented.
- 3Pankhurst, Q.; Jones, S.; Dobson, J. Applications of Magnetic Nanoparticles in Biomedicine: The Story so Far. J. Phys. D: Appl. Phys. 2016, 49, 501002, DOI: 10.1088/0022-3727/49/50/501002Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsFSru7fJ&md5=51a321039788a5ca12cb95d61af9bb99Applications of magnetic nanoparticles in biomedicine: the story so farPankhurst, Quentin; Jones, Stephen; Dobson, JonJournal of Physics D: Applied Physics (2016), 49 (50), 501002/1-501002/2CODEN: JPAPBE; ISSN:0022-3727. (IOP Publishing Ltd.)There is no expanded citation for this reference.
- 4Ling, D.; Lee, N.; Hyeon, T. Chemical Synthesis and Assembly of Uniformly Sized Iron Oxide Nanoparticles for Medical Applications. Acc. Chem. Res. 2015, 48, 1276– 1285, DOI: 10.1021/acs.accounts.5b00038Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXntlCmtb4%253D&md5=5623cdbf613bc8c00ae0725ec6f2b316Chemical Synthesis and Assembly of Uniformly Sized Iron Oxide Nanoparticles for Medical ApplicationsLing, Daishun; Lee, Nohyun; Hyeon, TaeghwanAccounts of Chemical Research (2015), 48 (5), 1276-1285CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. Magnetic iron oxide nanoparticles have been extensively investigated for their various biomedical applications including diagnostic imaging, biol. sensing, drug, cell, and gene delivery, and cell tracking. Recent advances in the designed synthesis and assembly of uniformly sized iron oxide nanoparticles have brought innovation in the field of nanomedicine. This Account provides a review on the recent progresses in the controlled synthesis and assembly of uniformly sized iron oxide nanoparticles for medical applications. In particular, it focuses on three topics: stringent control of particle size during synthesis via the "heat-up" process, surface modification for the high stability and biocompatibility of the nanoparticles for diagnostic purposes, and assembly of the nanoparticles within polymers or mesoporous silica matrixes for theranostic applications. Using extremely small 3 nm sized iron oxide nanoparticles (ESION), a new nontoxic T1 MRI contrast agent was realized for high-resoln. MRI of blood vessels down to 0.2 mm. Ferrimagnetic iron oxide nanoparticles (FION) that are larger than 20 nm exhibit extremely large magnetization and coercivity values. The cells labeled with FIONs showed very high T2 contrast effect so that even a single cell can be readily imaged. Designed assembly of iron oxide nanoparticles with mesoporous silica and polymers was conducted to fabricate multifunctional nanoparticles for theranostic applications. Mesoporous silica nanoparticles are excellent scaffolds for iron oxide nanoparticles, providing magnetic resonance and fluorescence imaging modalities as well as the functionality of the drug delivery vehicle. Polymeric ligands could be designed to respond to various biol. stimuli such as pH, temp., and enzymic activity. For example, we fabricated tumor pH-sensitive magnetic nanogrenades (termed PMNs) composed of self-assembled iron oxide nanoparticles and pH-responsive ligands. They were utilized to visualize small tumors (<3 mm) via pH-responsive T1 MRI and fluorescence imaging. Also, superior photodynamic therapeutic efficacy in highly drug-resistant heterogeneous tumors was obsd. We expect that these multifunctional and bioresponsive nanoplatforms based on uniformly sized iron oxide nanoparticles will provide more unique theranostic approaches in clin. uses.
- 5Giljohann, D. A.; Seferos, D. S.; Daniel, W. L.; Massich, M. D.; Patel, P. C.; Mirkin, C. A. Gold Nanoparticles for Biology and Medicine. Angew. Chem., Int. Ed. 2010, 49, 3280– 3294, DOI: 10.1002/anie.200904359Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXlt1GlsbY%253D&md5=0809d2a7d09124a8975d5ea77e679442Gold Nanoparticles for Biology and MedicineGiljohann, David A.; Seferos, Dwight S.; Daniel, Weston L.; Massich, Matthew D.; Patel, Pinal C.; Mirkin, Chad A.Angewandte Chemie, International Edition (2010), 49 (19), 3280-3294CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Gold colloids have fascinated scientists for over a century and are now heavily utilized in chem., biol., engineering, and medicine. Today these materials can be synthesized reproducibly, modified with seemingly limitless chem. functional groups, and, in certain cases, characterized with at.-level precision. This Review highlights recent advances in the synthesis, bioconjugation, and cellular uses of gold nanoconjugates. There are now many examples of highly sensitive and selective assays based upon gold nanoconjugates. In recent years, focus has turned to therapeutic possibilities for such materials. Structures which behave as gene-regulating agents, drug carriers, imaging agents, and photoresponsive therapeutics have been developed and studied in the context of cells and many debilitating diseases. These structures are not simply chosen as alternatives to mol.-based systems, but rather for their new phys. and chem. properties, which confer substantive advantages in cellular and medical applications.
- 6Alivisatos, A. P.; Johnsson, K. P.; Peng, X. G.; Wilson, T. E.; Loweth, C. J.; Bruchez, M. P.; Schultz, P. G. Organization of ’Nanocrystal Molecules’ Using DNA. Nature 1996, 382, 609– 611, DOI: 10.1038/382609a0Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XltVWqsrY%253D&md5=27e1eb981a0cfd51907eff5a5a105f50Organization of 'nanocrystal molecules' using DNAAlivisatos, A. Paul; Johnsson, Kai P.; Peng, Xiaogang; Wilson, Troy E.; Loweth, Colin J.; Bruchez, Marcel P., Jr.; Schultz, Peter G.Nature (London) (1996), 382 (6592), 609-611CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)The authors describe a strategy for the synthesis of 'nanocrystal mols.', in which discrete nos. of Au nanocrystals are organized into spatially defined structures based on Watson-Crick base-pairing interactions. The authors attach single-stranded DNA oligonucleotides of defined length and sequence to individual nanocrystals, and these assemble into dimers and trimers on addn. of a complementary single-stranded DNA template. The authors anticipate that this approach should allow the construction of more complex two- and three-dimensional assemblies.
- 7Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. A DNA-Based Method for Rationally Assembling Nanoparticles Into Macroscopic Materials. Nature 1996, 382, 607– 609, DOI: 10.1038/382607a0Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XltVWqsrk%253D&md5=11128671758a23f01bf6c8d3b20b3921A DNA-based method for rationally assembling nanoparticles into macroscopic materialsMirkin, Chad A.; Letsinger, Robert L.; Mucic, Robert C.; Storhoff, James J.Nature (London) (1996), 382 (6592), 607-609CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)Colloidal particles of metals and semiconductors have potentially useful optical, optoelectronic and material properties that derive from their small (nanoscopic) size. These properties might lead to applications including chem. sensors, spectroscopic enhancers, quantum dot and nanostructure fabrication, and microimaging methods. A great deal of control can now be exercised over the chem. compn., size and polydispersity of colloidal particles, and many methods have been developed for assembling them into useful aggregates and materials. Here we describe a method for assembling colloidal gold nanoparticles rationally and reversibly into macroscopic aggregates. The method involves attaching to the surfaces of two batches of 13-nm gold particles non-complementary DNA oligonucleotides capped with thiol groups, which bind to gold. When we add to the soln. an oligonucleotide duplex with 'sticky ends' that are complementary to the two grafted sequences, the nanoparticles self-assemble into aggregates. This assembly process can be reversed by thermal denaturation. This strategy should now make it possible to tailor the optical, electronic and structural properties of the colloidal aggregates by using the specificity of DNA interactions to direct the interactions between particles of different size and compn.
- 8Cutler, J. I.; Auyeung, E.; Mirkin, C. A. Spherical Nucleic Acids. J. Am. Chem. Soc. 2012, 134, 1376– 1391, DOI: 10.1021/ja209351uGoogle Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XktVKlsQ%253D%253D&md5=0ccb9d7fab59aee9e0984c4e7d905d37Spherical Nucleic AcidsCutler, Joshua I.; Auyeung, Evelyn; Mirkin, Chad A.Journal of the American Chemical Society (2012), 134 (3), 1376-1391CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A review. A historical perspective of the development of spherical nucleic acid (SNA) conjugates and other three-dimensional nucleic acid nanostructures is provided. This Perspective details the synthetic methods for prepg. them, followed by a discussion of their unique properties and theor. and exptl. models for understanding them. Important examples of technol. advances made possible by their fundamental properties spanning the fields of chem., mol. diagnostics, gene regulation, medicine, and materials science are also presented.
- 9Hurst, S. J.; Hill, H. D.; Mirkin, C. A. ″Three-Dimensional Hybridization″ With Polyvalent DNA-Gold Nanoparticle Conjugates. J. Am. Chem. Soc. 2008, 130, 12192– 12200, DOI: 10.1021/ja804266jGoogle ScholarThere is no corresponding record for this reference.
- 10Jones, M. R.; Macfarlane, R. J.; Lee, B.; Zhang, J. A.; Young, K. L.; Senesi, A. J.; Mirkin, C. A. DNA-Nanoparticle Superlattices Formed From Anisotropic Building Blocks. Nat. Mater. 2010, 9, 913– 917, DOI: 10.1038/nmat2870Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtlWkt7zK&md5=6e6c918813e2bd715c9a3c44203d38ddDNA-nanoparticle superlattices formed from anisotropic building blocksJones, Matthew R.; MacFarlane, Robert J.; Lee, Byeongdu; Zhang, Jian; Young, Kaylie L.; Senesi, Andrew J.; Mirkin, Chad A.Nature Materials (2010), 9 (11), 913-917CODEN: NMAACR; ISSN:1476-1122. (Nature Publishing Group)Directional bonding interactions in solid-state at. lattices dictate the unique symmetries of at. crystals, resulting in a diverse and complex assortment of three-dimensional structures that exhibit a wide variety of material properties. Methods to create analogous nanoparticle superlattices are beginning to be realized, but the concept of anisotropy is still largely underdeveloped in most particle assembly schemes. Some examples provide interesting methods to take advantage of anisotropic effects, but most are able to make only small clusters or lattices that are limited in crystallinity and esp. in lattice parameter programmability. Anisotropic nanoparticles can be used to impart directional bonding interactions on the nanoscale, both through face-selective functionalization of the particle with recognition elements to introduce the concept of valency, and through anisotropic interactions resulting from particle shape. The authors examine the concept of inherent shape-directed crystn. in the context of DNA-mediated nanoparticle assembly. Importantly, the anisotropy of these particles can be used to synthesize 1-, two- and three-dimensional structures that cannot be made through the assembly of spherical particles.
- 11Park, S. Y.; Lytton-Jean, A. K. R.; Lee, B.; Weigand, S.; Schatz, G. C.; Mirkin, C. A. DNA-Programmable Nanoparticle Crystallization. Nature 2008, 451, 553– 556, DOI: 10.1038/nature06508Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhs1ent7g%253D&md5=b5c8c1ab8b7f43d32793560e4869ad2fDNA-programmable nanoparticle crystallizationPark, Sung Yong; Lytton-Jean, Abigail K. R.; Lee, Byeongdu; Weigand, Steven; Schatz, George C.; Mirkin, Chad A.Nature (London, United Kingdom) (2008), 451 (7178), 553-556CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)It was first shown more than ten years ago that DNA oligonucleotides can be attached to gold nanoparticles rationally to direct the formation of larger assemblies. Since then, oligonucleotide-functionalized nanoparticles have been developed into powerful diagnostic tools for nucleic acids and proteins, and into intracellular probess and gene regulators. In contrast, the conceptually simple yet powerful idea that functionalized nanoparticles might serve as basic building blocks that can be rationally assembled through programmable base-pairing interactions into highly ordered macroscopic materials remains poorly developed. So far, the approach has mainly resulted in polymn., with modest control over the placement of, the periodicity in, and the distance between particles within the assembled material. That is, most of the materials obtained thus far are best classified as amorphous polymers, although a few examples of colloidal crystal formation exist. Here, we demonstrate that DNA can be used to control the crystn. of nanoparticle-oligonucleotide conjugates to the extent that different DNA sequences guide the assembly of the same type of inorg. nanoparticle into different cryst. states. We show that the choice of DNA sequences attached to the nanoparticle building blocks, the DNA linking mols. and the absence or presence of a non-bonding single-base flexor can be adjusted so that gold nanoparticles assemble into micrometer-sized face-centered-cubic or body-centered-cubic crystal structures. Our findings thus clearly demonstrate that synthetically programmable colloidal crystn. is possible, and that a single-component system can be directed to form different structures.
- 12Park, S. J.; Lazarides, A. A.; Storhoff, J. J.; Pesce, L.; Mirkin, C. A. The Structural Characterization of Oligonucleotide-Modified Gold Nanoparticle Networks Formed by DNA Hybridization. J. Phys. Chem. B 2004, 108, 12375– 12380, DOI: 10.1021/jp040242bGoogle Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXlvFGisLw%253D&md5=7c40303add3bf2ba27d47b79e4f9fda6The Structural Characterization of Oligonucleotide-Modified Gold Nanoparticle Networks Formed by DNA HybridizationPark, So-Jung; Lazarides, Anne A.; Storhoff, James J.; Pesce, Lorenzo; Mirkin, Chad A.Journal of Physical Chemistry B (2004), 108 (33), 12375-12380CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)The structural properties of DNA-linked gold nanoparticle materials were examd. using synchrotron small-angle X-ray scattering (SAXS). The materials are composed of 12 or 19 nm diam. gold particles modified with 3' or 5' alkylthiol-capped 12-base oligonucleotides and linked with complementary oligonucleotides. Structure factors were derived from scattering intensities, and nearest-neighbor distances were detd. from the primary peak in the pair distance distribution functions. The sepn. between particles was found to increase linearly with DNA linker length for 24, 48, and 72 base pair linkers. For assemblies formed in 0.3 M NaCl, 10 mM phosphate buffer soln., the increment in the interparticle distance was found to be 2.5 Å per base pair. Particle sepns. in assemblies at lower electrolyte concn. were larger, indicating that dielec. screening modulates the interactions. The effect of DNA sequence was studied with poly-adenine or poly-thymine spacer sequences incorporated between the alkylthiol and recognition sequences. The assemblies with poly-adenine spacer sequences showed significantly shorter particle sepns. than the assemblies involving poly-thymine spacers, a consequence of their different affinities for the gold surface. While the scattering data do not display evidence of long-range order, pair distance distribution functions indicate the presence of short-range order.
- 13Nykypanchuk, D.; Maye, M. M.; van der Lelie, D.; Gang, O. DNA-Guided Crystallization of Colloidal Nanoparticles. Nature 2008, 451, 549– 552, DOI: 10.1038/nature06560Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhs1ent7s%253D&md5=dba644c8f297d79a3a9cc5d1b64ad9c3DNA-guided crystallization of colloidal nanoparticlesNykypanchuk, Dmytro; Maye, Matthew M.; van der Lelie, Daniel; Gang, OlegNature (London, United Kingdom) (2008), 451 (7178), 549-552CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Many nanometer-size building blocks will readily assemble into macroscopic structures. If the process is accompanied by effective control over the interactions between the blocks and all entropic effects, then the resultant structures will be ordered with a precision hard to achieve with other fabrication methods. But it remains challenging to use self-assembly to design systems comprised of different types of building blocks - to realize novel magnetic, plasmonic and photonic metamaterials, for example. A conceptually simple idea for overcoming this problem is the use of 'encodable' interactions between building blocks; this can in principle be straightforwardly implemented using biomols. Strategies that use DNA programmability to control the placement of nanoparticles in one and two dimensions have indeed been demonstrated. However, theor. understanding of how to extend this approach to 3 dimensions is limited, and most expts. have yielded amorphous aggregates and only occasionally crystallites of close-packed micrometer-size particles. Here, the authors report the formation of 3-dimensional cryst. assemblies of gold nanoparticles mediated by interactions between complementary DNA mols. attached to the nanoparticles' surface. We find that the nanoparticle crystals form reversibly during heating and cooling cycles. Moreover, the body-centered-cubic lattice structure is temp.-tuneable and structurally open, with particles occupying only ∼4% of the unit cell vol. The authors expect that the DNA-mediated crystn. approach, and the insight into DNA design requirements it has provided, will facilitate both the creation of new classes of ordered multicomponent metamaterials and the exploration of the phase behavior of hybrid systems with addressable interactions.
- 14Harimech, P. K.; Gerrard, S. R.; El-Sagheer, A. H.; Brown, T.; Kanaras, A. G. Reversible Ligation of Programmed DNA-Gold Nanoparticle Assemblies. J. Am. Chem. Soc. 2015, 137, 9242– 9245, DOI: 10.1021/jacs.5b05683Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtF2ltr7J&md5=5f440a69ae6c73433d873ceeadbf4becReversible Ligation of Programmed DNA-Gold Nanoparticle AssembliesHarimech, Pascal K.; Gerrard, Simon R.; El-Sagheer, Afaf H.; Brown, Tom; Kanaras, Antonios G.Journal of the American Chemical Society (2015), 137 (29), 9242-9245CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We demonstrate a new method to reversibly crosslink DNA-nanoparticle dimers, trimers, and tetramers using light as an external stimulus. A DNA interstrand photo-crosslinking reaction is possible via ligation of a cyano-vinyl carbazole nucleoside with an opposite thymine when irradiated at 365 nm. This reaction results in nanoparticle assemblies that are not susceptible to DNA dehybridization conditions. The chem. bond between the two complementary DNA strands can be reversibly broken upon light irradn. at 312 nm. This is the first example of reversible ligation in DNA-nanoparticle assemblies using light and enables new developments in the field of programmed nanoparticle organization.
- 15Heuer-Jungemann, A.; Kirkwood, R.; El-Sagheer, A. H.; Brown, T.; Kanaras, A. G. Copper-Free Click Chemistry as an Emerging Tool for the Programmed Ligation of DNA-Functionalised Gold Nanoparticles. Nanoscale 2013, 5, 7209– 7212, DOI: 10.1039/c3nr02362aGoogle Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtFKgu7vI&md5=fc12ba5108d6bb99b25ee7f6efaed8d1Copper-free click chemistry as an emerging tool for the programmed ligation of DNA-functionalised gold nanoparticlesHeuer-Jungemann, Amelie; Kirkwood, Robert; El-Sagheer, Afaf H.; Brown, Tom; Kanaras, Antonios G.Nanoscale (2013), 5 (16), 7209-7212CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)We demonstrate a new method to program the ligation of single stranded DNA-modified gold nanoparticles using copper-free click chem. Gold nanoparticles functionalized with a discrete no. of 3'-azide or 5'-alkyne modified oligonucleotides, can be brought together via a splint strand and covalently 'clicked', in a simple one-pot reaction. This new approach to the assembly of gold nanoparticles is inherently advantageous in comparison to the traditional enzymic ligation. The chem. ligation is specific and takes place at room temp. by simply mixing the particles without the need for special enzymic conditions. The yield of 'clicked' nanoparticles can be as high as 92%. The ease of the copper-free, click-ligation' method allows for its universal applicability and opens up new avenues in programmed nanoparticle organization.
- 16Hurst, S. J.; Lytton-Jean, A. K. R.; Mirkin, C. A. Maximizing DNA Loading on a Range of Gold Nanoparticle Sizes. Anal. Chem. 2006, 78, 8313– 8318, DOI: 10.1021/ac0613582Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFyhtrfO&md5=1b1f5ac1386517068d1252b850112211Maximizing DNA Loading on a Range of Gold Nanoparticle SizesHurst, Sarah J.; Lytton-Jean, Abigail K. R.; Mirkin, Chad A.Analytical Chemistry (2006), 78 (24), 8313-8318CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)The authors have investigated the variables that influence DNA coverage on gold nanoparticles. The effects of salt concn., spacer compn., nanoparticle size, and degree of sonication have been evaluated. Maximum loading was obtained by salt aging the nanoparticles to ∼0.7 M NaCl in the presence of DNA contg. a poly(ethylene glycol) spacer. In addn., DNA loading was substantially increased by sonicating the nanoparticles during the surface loading process. Last, nanoparticles up to 250 nm in diam. were found have ∼2 orders of magnitude higher DNA loading than smaller (13-30 nm) nanoparticles, a consequence of their larger surface area. Stable large particles are attractive for a variety of biodiagnostic assays.
- 17Choi, C. H. J.; Hao, L.; Narayan, S. P.; Auyeung, E.; Mirkin, C. A. Mechanism for the Endocytosis of Spherical Nucleic Acid Nanoparticle Conjugates. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 7625– 7630, DOI: 10.1073/pnas.1305804110Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXptFGrt7k%253D&md5=f0755c5fa2b33b5ca167ef14c6394b27Mechanism for the endocytosis of spherical nucleic acid nanoparticle conjugatesChoi, Chung Hang J.; Hao, Liangliang; Narayan, Suguna P.; Auyeung, Evelyn; Mirkin, Chad A.Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (19), 7625-7630, S7625/1-S7625/7CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Intracellular delivery of nucleic acids as gene regulation agents typically requires the use of cationic carriers or viral vectors, yet issues related to cellular toxicity or immune responses hamper their attractiveness as therapeutic candidates. The discovery that spherical nucleic acids (SNAs), polyanionic structures comprised of densely packed, highly oriented oligonucleotides covalently attached to the surface of nanoparticles, can effectively enter more than 50 different cell types presents a potential strategy for overcoming the limitations of conventional transfection agents. Unfortunately, little is known about the mechanism of endocytosis of SNAs, including the pathway of entry and specific proteins involved. Here, we demonstrate that the rapid cellular uptake kinetics and intracellular transport of SNAs stem from the arrangement of oligonucleotides into a 3D architecture, which supports their targeting of class A scavenger receptors and endocytosis via a lipid-raft-dependent, caveolae-mediated pathway. These results reinforce the notion that SNAs can serve as therapeutic payloads and targeting structures to engage biol. pathways not readily accessible with linear oligonucleotides.
- 18Wu, X. A.; Choi, C. H. J.; Zhang, C.; Hao, L.; Mirkin, C. A. Intracellular Fate of Spherical Nucleic Acid Nanoparticle Conjugates. J. Am. Chem. Soc. 2014, 136, 7726– 7733, DOI: 10.1021/ja503010aGoogle Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXotFOhs7k%253D&md5=b2e703b091edc7eb39931e96ba5c4b68Intracellular Fate of Spherical Nucleic Acid Nanoparticle ConjugatesWu, Xiaochen A.; Choi, Chung Hang J.; Zhang, Chuan; Hao, Liangliang; Mirkin, Chad A.Journal of the American Chemical Society (2014), 136 (21), 7726-7733CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Spherical nucleic acid (SNA) nanoparticle conjugates are a class of bionanomaterials that are extremely potent in many biomedical applications. Their unique ability to enter multiple mammalian cell types as single-entity agents arises from their novel three-dimensional architecture, which consists of a dense shell of highly oriented oligonucleotides chem. attached typically to a gold nanoparticle core. This architecture allows SNAs to engage certain cell surface receptors to facilitate entry. Here, we report studies aimed at detg. the intracellular fate of SNAs and the trafficking events that occur inside C166 mouse endothelial cells after cellular entry. We show that SNAs traffic through the endocytic pathway into late endosomes and reside there for up to 24 h after incubation. Disassembly of oligonucleotides from the nanoparticle core is obsd. 16 h after cellular entry, most likely due to degrdn. by enzymes such as DNase II localized in late endosomes. Our observations point to these events being likely independent of core compn. and treatment conditions, and they do not seem to be particularly dependent upon oligonucleotide sequence. Significantly and surprisingly, the SNAs do not enter the lysosomes under the conditions studied. To independently track the fate of the particle core and the fluorophore-labeled oligonucleotides that comprise its shell, we synthesized a novel class of quantum dot SNAs to det. that as the SNA structures are broken down over the 24 h time course of the expt., the oligonucleotide fragments are recycled out of the cell while the nanoparticle core is not. This mechanistic insight points to the importance of designing and synthesizing next-generation SNAs that can bypass the degrdn. bottleneck imposed by their residency in late endosomes, and it also suggests that such structures might be extremely useful for endosomal signaling pathways by engaging receptors that are localized within the endosome.
- 19Giljohann, D. A.; Seferos, D. S.; Patel, P. C.; Millstone, J. E.; Rosi, N. L.; Mirkin, C. A. Oligonucleotide Loading Determines Cellular Uptake of DNA-Modified Gold Nanoparticles. Nano Lett. 2007, 7, 3818– 3821, DOI: 10.1021/nl072471qGoogle Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht1yrurvM&md5=46417b236e7d7075a484007a474f9acbOligonucleotide Loading Determines Cellular Uptake of DNA-Modified Gold NanoparticlesGiljohann, David A.; Seferos, Dwight S.; Patel, Pinal C.; Millstone, Jill E.; Rosi, Nathaniel L.; Mirkin, Chad A.Nano Letters (2007), 7 (12), 3818-3821CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The cellular internalization of oligonucleotide-modified nanoparticles is investigated. Uptake is dependent on the d. of the oligonucleotide loading on the surface of the particles, where higher densities lead to greater uptake. Densely functionalized nanoparticles adsorb a large no. of proteins on the nanoparticle surface. Nanoparticle uptake is greatest where a large no. of proteins are assocd. with the particle.
- 20Patel, P. C.; Giljohann, D. A.; Daniel, W. L.; Zheng, D.; Prigodich, A. E.; Mirkin, C. A. Scavenger Receptors Mediate Cellular Uptake of Polyvalent Oligonucleotide-Functionalized Gold Nanoparticles. Bioconjugate Chem. 2010, 21, 2250– 2256, DOI: 10.1021/bc1002423Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVSisbbF&md5=d907b2d964481128d4888696ef1dc22cScavenger Receptors Mediate Cellular Uptake of Polyvalent Oligonucleotide-Functionalized Gold NanoparticlesPatel, Pinal C.; Giljohann, David A.; Daniel, Weston L.; Zheng, Dan; Prigodich, Andrew E.; Mirkin, Chad A.Bioconjugate Chemistry (2010), 21 (12), 2250-2256CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)Mammalian cells have been shown to internalize oligonucleotide-functionalized gold nanoparticles (DNA-Au NPs or siRNA-Au NPs) without the aid of auxiliary transfection agents and use them to initiate an antisense or RNAi response. Previous studies have shown that the dense monolayer of oligonucleotides on the nanoparticle leads to the adsorption of serum proteins and facilitates cellular uptake. Here, we show that serum proteins generally act to inhibit cellular uptake of DNA-Au NPs. We identify the pathway for DNA-Au NP entry in HeLa cells. Biochem. analyses indicate that DNA-Au NPs are taken up by a process involving receptor-mediated endocytosis. Evidence shows that DNA-Au NP entry is primarily mediated by scavenger receptors, a class of pattern-recognition receptors. This uptake mechanism appears to be conserved across species, as blocking the same receptors in mouse cells also disrupted DNA-Au NP entry. Polyvalent nanoparticles functionalized with siRNA are shown to enter through the same pathway. Thus, scavenger receptors are required for cellular uptake of polyvalent oligonucleotide functionalized nanoparticles.
- 21Seferos, D. S.; Prigodich, A. E.; Giljohann, D. A.; Patel, P. C.; Mirkin, C. A. Polyvalent DNA Nanoparticle Conjugates Stabilize Nucleic Acids. Nano Lett. 2009, 9, 308– 311, DOI: 10.1021/nl802958fGoogle Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsFantbfL&md5=9c13243d65bae4af040f17b8d828444fPolyvalent DNA Nanoparticle Conjugates Stabilize Nucleic AcidsSeferos, Dwight S.; Prigodich, Andrew E.; Giljohann, David A.; Patel, Pinal C.; Mirkin, Chad A.Nano Letters (2009), 9 (1), 308-311CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Polyvalent oligonucleotide gold nanoparticle conjugates have unique fundamental properties including distance-dependent plasmon coupling, enhanced binding affinity, and the ability to enter cells and resist enzymic degrdn. Stability in the presence of enzymes is a key consideration for therapeutic uses; however the manner and mechanism by which such nanoparticles are able to resist enzymic degrdn. is unknown. Here, we quantify the enhanced stability of polyvalent gold oligonucleotide nanoparticle conjugates with respect to enzyme-catalyzed hydrolysis of DNA and present evidence that the neg. charged surfaces of the nanoparticles and resultant high local salt concns. are responsible for enhanced stability.
- 22Heuer-Jungemann, A.; Harimech, P. K.; Brown, T.; Kanaras, A. G. Gold Nanoparticles and Fluorescently-Labelled DNA as a Platform for Biological Sensing. Nanoscale 2013, 5, 9503– 9510, DOI: 10.1039/c3nr03707jGoogle Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsFaltrbP&md5=1cfc1c9836f3c35d00cf14180c621116Gold nanoparticles and fluorescently-labelled DNA as a platform for biological sensingHeuer-Jungemann, Amelie; Harimech, Pascal K.; Brown, Tom; Kanaras, Antonios G.Nanoscale (2013), 5 (20), 9503-9510CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)A review. In the past decade gold nanoparticle-nucleic acid conjugates became progressively important for biomedical applications. Fluorophores attached to nucleic acid-gold nanoparticle conjugates have opened up a new era of biol. sensing. The most promising advancement in this field was the invention of the so-called nano-flare' systems. These systems are capable of detecting specific endocellular targets such as mRNAs, microRNAs or small mols. in real time. In this minireview, we discuss the current progress in the field of DNA-nanoparticles as sensors, their properties, stability, cellular uptake and cytotoxicity.
- 23Seferos, D. S.; Giljohann, D. A.; Hill, H. D.; Prigodich, A. E.; Mirkin, C. A. Nano-flares: Probes for Transfection and mRNA Detection in Living Cells. J. Am. Chem. Soc. 2007, 129, 15477– 15479, DOI: 10.1021/ja0776529Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlKru7jE&md5=71e766b235655781c09ccfc331b37762Nano-Flares: Probes for Transfection and mRNA Detection in Living CellsSeferos, Dwight S.; Giljohann, David A.; Hill, Haley D.; Prigodich, Andrew E.; Mirkin, Chad A.Journal of the American Chemical Society (2007), 129 (50), 15477-15479CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We demonstrate that novel oligonucleotide-modified gold nanoparticle probes hybridized to fluorophore-labeled complements can be used as both transfection agents and cellular "nano-flares" for detecting mRNA in living cells. Nano-flares take advantage of the highly efficient fluorescence quenching properties of gold, cellular uptake of oligonucleotide nanoparticle conjugates without the use of transfection agents, and the enzymic stability of such conjugates, thus overcoming many of the challenges to creating sensitive and effective intracellular probes. Nano-flares exhibit high signaling, have low background fluorescence, and are sensitive to changes in the no. of RNA transcripts present in cells.
- 24Xue, J.; Shan, L.; Chen, H.; Li, Y.; Zhu, H.; Deng, D.; Qian, Z.; Achilefu, S.; Gu, Y. Visual Detection of STAT5B Gene Expression in Living Cell Using the Hairpin DNA Modified Gold Nanoparticle Beacon. Biosens. Bioelectron. 2013, 41, 71– 77, DOI: 10.1016/j.bios.2012.06.062Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsF2nurnN&md5=3cabf394d58f9bd1d28f2193131d4399Visual detection of STAT5B gene expression in living cell using the hairpin DNA modified gold nanoparticle beaconXue, Jianpeng; Shan, Lingling; Chen, Haiyan; Li, Yang; Zhu, Hongyan; Deng, Dawei; Qian, Zhiyu; Achilefu, Samuel; Gu, YueqingBiosensors & Bioelectronics (2013), 41 (), 71-77CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)Signal transducer and activator of transcription 5B (STAT5B) is an important protein in JAK-STAT signaling pathway that is responsible for the metastasis and proliferation of tumor cells. Detn. of the STAT5B mRNA relating to the STAT5B expression provides insight into the mechanism of tumor progression. In this study, we designed and used a special hairpin DNA for human STAT5B mRNA to functionalize gold nanoparticles, which served as a beacon for detecting human STAT5B expression. Up to 90% quenching efficiency was achieved. Upon hybridizing with the target mRNA, the hairpin DNA modified gold nanoparticle beacons (hDAuNP beacons) release the fluorophores attached at 5' end of the oligonucleotide sequence. The fluorescence properties of the beacon before and after the hybridization with the complementary DNA were confirmed in vitro. The stability of hDAuNP beacons against degrdn. by DNase I and GSH indicated that the prepd. beacon is stable inside cells. The detected fluorescence in MCF-7 cancer cells correlates with the specific STAT5B mRNA expression, which is consistent with the result from PCR measurement. Fluorescence microscopy showed that the hDAuNP beacons internalized in cells without using transfection agents, with intracellular distribution in the cytoplasm rather than the nucleus. The results demonstrated that this beacon could directly provide quant. measurement of the intracellular STAT5B mRNA in living cells. Compared to the previous approaches, this beacon has advantages of higher target to background ratio of detection and an increased resistance to nuclease degrdn. The strategy reported in this study is a promising approach for the intracellular measurement of RNA or protein expression in living cells, and has great potential in the study of drug screening and discovery.
- 25Shi, J.; Zhou, M.; Gong, A. H.; Li, Q. J.; Wu, Q.; Cheng, G. J.; Yang, M. Y.; Sun, Y. C. Fluorescence Lifetime Imaging of Nanoflares for mRNA Detection in Living Cells. Anal. Chem. 2016, 88, 1979– 1983, DOI: 10.1021/acs.analchem.5b03689Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1KntLw%253D&md5=6344f2d0c3e81690e12cffdf6c60c6a7Fluorescence Lifetime Imaging of Nanoflares for mRNA Detection in Living CellsShi, Jing; Zhou, Ming; Gong, Aihua; Li, Qijun; Wu, Qian; Cheng, Gary J.; Yang, Mingyang; Sun, YaochengAnalytical Chemistry (Washington, DC, United States) (2016), 88 (4), 1979-1983CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)The expression level of tumor-related mRNA can reveal significant information about tumor progression and prognosis, so specific mRNA in cells provides an important approach for biol. and disease studies. Here, fluorescence lifetime imaging of nanoflares in living cells was first employed to detect specific intracellular mRNA. We characterized the lifetime changes of the prepd. nanoflares before and after the treatment of target mRNA and also compared the results with those of fluorescence intensity-based measurements both intracellularly and extracellularly. The nanoflares released the Cy5-modified oligonucleotides and bound to the targets, resulting in a fluorescence lifetime lengthening. This work puts forward another dimension of detecting specific mRNA in cells and can also open new ways for detection of many other biomols.
- 26Halo, T. L.; McMahon, K. M.; Angeloni, N. L.; Xu, Y.; Wang, W.; Chinen, A. B.; Malin, D.; Strekalova, E.; Cryns, V. L.; Cheng, C.; Mirkin, C. A.; Thaxton, C. S. NanoFlares for the Detection,Isolation, and Culture of Live Tumor Cells from Human Blood. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 17104– 17109, DOI: 10.1073/pnas.1418637111Google ScholarThere is no corresponding record for this reference.
- 27Prigodich, A. E.; Seferos, D. S.; Massich, M. D.; Giljohann, D. A.; Lane, B. C.; Mirkin, C. A. Nano-flares for mRNA Regulation and Detection. ACS Nano 2009, 3, 2147– 2152, DOI: 10.1021/nn9003814Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXotl2ht7g%253D&md5=85f5a40b73e9de5bb5ce944891f8a987Nano-flares for mRNA regulation and detectionPrigodich, Andrew E.; Seferos, Dwight S.; Massich, Matthew D.; Giljohann, David A.; Lane, Brandon C.; Mirkin, Chad A.ACS Nano (2009), 3 (8), 2147-2152CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)We build off the previously described concept of a nanoflare to develop an oligonucleotide gold nanoparticle conjugate that is capable of both detecting and regulating intracellular levels of mRNA. We characterize the binding rate and specificity of these materials using survivin, a gene assocd. with the diagnosis and treatment of cancer, as a target. The nanoconjugate enters cells and binds mRNA, thereby decreasing the relative abundance of mRNA in a dose- and sequence-dependent manner, resulting in a fluorescent response. This represents the first demonstration of a single material capable of both mRNA regulation and detection. Further, we investigate the intracellular biochem. of the nanoconjugate, elucidating its mechanism of gene regulation. This work is important to the study of biol. active nanomaterials such as the nanoflare and is a first step toward the development of an mRNA responsive "theranostic".
- 28McClellan, S.; Slamecka, J.; Howze, P.; Thompson, L.; Finan, M.; Rocconi, R.; Owen, L. mRNA Detection in Living Cells: A Next Generation Cancer Stem Cell Identification Technique. Methods 2015, 82, 47– 54, DOI: 10.1016/j.ymeth.2015.04.022Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXntlWntrw%253D&md5=2fc899ba37e9f56c44af074a676b7789mRNA detection in living cells: A next generation cancer stem cell identification techniqueMcClellan, Steven; Slamecka, Jaroslav; Howze, Patrick; Thompson, Lee; Finan, Michael; Rocconi, Rodney; Owen, LaurieMethods (Amsterdam, Netherlands) (2015), 82 (), 47-54CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)Cancer stem cells (CSC) are a distinct subpopulation within a tumor shown to drive tumor progression, metastasis, and recurrence. A review of the literature reveals poor consensus, with the use of a wide variety of surface markers and functional assays to identify and isolate cancer stem cells. Utilizing a novel technol. that enables live-cell mRNA quantitation, we have demonstrated the ability to identify and sort viable CSC based on markers assocd. with stemness in pluripotent cells. Fresh tumor samples from a variety of cancer types were examd. by flow cytometry for Nanog expression. Levels of CSC detected ranged from 6% to 19%. This method of CSC detection was cross-validated with other commonly used surface markers with good correlation. Matrigel invasion assays confirmed that CSC isolated using this method are both highly motile and invasive. This approach simplifies the process of identifying viable CSC from fresh tumor tissue, providing a level of accuracy not previously available. This method may also provide a valuable tool for screening and validating new CSC biomarkers.
- 29Seftor, E. A.; Seftor, R. E. B.; Weldon, D. S.; Kirsammer, G. T.; Margaryan, N. V.; Gilgur, A.; Hendrix, M. J. C. Melanoma Tumor Cell Heterogeneity: A Molecular Approach to Study Subpopulations Expressing the Embryonic Morphogen Nodal. Semin. Oncol. 2014, 41, 259– 266, DOI: 10.1053/j.seminoncol.2014.02.001Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXns1Cltbs%253D&md5=9b6319f023f4a4d3b573b0d05883b7a5Melanoma Tumor Cell Heterogeneity: A Molecular Approach to Study Subpopulations Expressing the Embryonic Morphogen NodalSeftor, Elisabeth A.; Seftor, Richard E. B.; Weldon, Don S.; Kirsammer, Gina T.; Margaryan, Naira V.; Gilgur, Alina; Hendrix, Mary J. C.Seminars in Oncology (2014), 41 (2), 259-266CODEN: SOLGAV; ISSN:0093-7754. (Elsevier Inc.)A review. As the frequency of melanoma increases, current treatment strategies are struggling to significantly impact patient survival. One of the crit. issues in designing efficient therapies is understanding the compn. of heterogeneous melanoma tumors in order to target cancer stem cells (CSCs) and drug-resistant subpopulations. In this review, we summarize recent findings pertinent to the reemergence of the embryonic Nodal signaling pathway in melanoma and its significance as a prognostic biomarker and therapeutic target. In addn., we offer a novel mol. approach to studying the functional relevance of Nodal-expressing subpopulations and their CSC phenotype.
- 30Li, B. J.; Menzel, U.; Loebel, C.; Schmal, H.; Alini, M.; Stoddart, M. J. Monitoring Live Human Mesenchymal Stromal Cell Differentiation and Subsequent Selection Using Fluorescent RNA-Based Probes. Sci. Rep. 2016, 6, 26014, DOI: 10.1038/srep26014Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xotlemtb8%253D&md5=cf957f47f61d6bb444b3a32976b98ecaMonitoring live human mesenchymal stromal cell differentiation and subsequent selection using fluorescent RNA-based probesLi, Bojun; Menzel, Ursula; Loebel, Claudia; Schmal, Hagen; Alini, Mauro; Stoddart, Martin J.Scientific Reports (2016), 6 (), 26014CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)Investigating mesenchymal stromal cell differentiation requires time and multiple samples due to destructive endpoint assays. Osteogenesis of human bone marrow derived mesenchymal stromal cells (hBMSCs) has been widely studied for bone tissue engineering. Recent studies show that the osteogenic differentiation of hBMSCs can be assessed by quantifying the ratio of two important transcription factors (Runx2/Sox9). We demonstrate a method to observe mRNA expression of two genes in individual live cells using fluorescent probes specific for Runx2 and Sox9 mRNA. The changes of mRNA expression in cells can be obsd. in a non-destructive manner. In addn., the osteogenic hBMSCs can be prospectively identified and obtained based on the relative intracellular fluorescence of Sox9 in relation to Runx2 using fluorescence activated cell sorting. Relatively homogeneous cell populations with high osteogenic potential can be isolated from the original heterogeneous osteogenically induced hBMSCs within the first week of induction. This offers a more detailed anal. of the effectiveness of new therapeutics both at the individual cell level and the response of the population as a whole. By identifying and isolating differentiating cells at early time points, prospective anal. of differentiation is also possible, which will lead to a greater understanding of MSC differentiation.
- 31Lahm, H.; Doppler, S.; Dressen, M.; Werner, A.; Adamczyk, K.; Schrambke, D.; Brade, T.; Laugwitz, K. L.; Deutsch, M. A.; Schiemann, M.; Lange, R.; Moretti, A.; Krane, M. Live Fluorescent RNA-Based Detection of Pluripotency Gene Expression in Embryonic and Induced Pluripotent Stem Cells of Different Species. Stem Cells 2015, 33, 392– 402, DOI: 10.1002/stem.1872Google ScholarThere is no corresponding record for this reference.
- 32Prigodich, A. E.; Randeria, P. S.; Briley, W. E.; Kim, N. J.; Daniel, W. L.; Giljohann, D. A.; Mirkin, C. A. Multiplexed Nanoflares: mRNA Detection in Live Cells. Anal. Chem. 2012, 84, 2062– 2066, DOI: 10.1021/ac202648wGoogle Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsVWitbk%253D&md5=8a1084688f99c699ca49314dbc328e66Multiplexed Nanoflares: mRNA Detection in Live CellsPrigodich, Andrew E.; Randeria, Pratik S.; Briley, William E.; Kim, Nathaniel J.; Daniel, Weston L.; Giljohann, David A.; Mirkin, Chad A.Analytical Chemistry (Washington, DC, United States) (2012), 84 (4), 2062-2066CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)We report the development of the multiplexed nanoflare, a nanoparticle agent that is capable of simultaneously detecting two distinct mRNA targets inside a living cell. These probes are spherical nucleic acid (SNA) gold nanoparticle (Au NP) conjugates consisting of densely packed and highly oriented oligonucleotide sequences, many of which are hybridized to a reporter with a distinct fluorophore label and each complementary to its corresponding mRNA target. When multiplexed nanoflares are exposed to their targets, they provide a sequence specific signal in both extra- and intracellular environments. Importantly, one of the targets can be used as an internal control, improving detection by accounting for cell-to-cell variations in nanoparticle uptake and background. Compared to single-component nanoflares, these structures allow one to det. more precisely relative mRNA levels in individual cells, improving cell sorting and quantification.
- 33Pan, W.; Zhang, T.; Yang, H.; Diao, W.; Li, N.; Tang, B. Multiplexed Detection and Imaging of Intracellular mRNAs Using a Four-Color Nanoprobe. Anal. Chem. 2013, 85, 10581– 10588, DOI: 10.1021/ac402700sGoogle Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsFGkurjF&md5=a3172b1a65d1a6c5cc252783acc5ff37Multiplexed Detection and Imaging of Intracellular mRNAs Using a Four-Color NanoprobePan, Wei; Zhang, Tingting; Yang, Huijun; Diao, Wei; Li, Na; Tang, BoAnalytical Chemistry (Washington, DC, United States) (2013), 85 (21), 10581-10588CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Simultaneous detection and imaging of multiple intracellular biomarkers hold great promise for early cancer detection. Here, we introduce a four-color nanoprobe that can simultaneously detect and image four types of mRNAs in living cells. The nanoprobe composed of gold nanoparticles functionalized with a dense shell of mol. beacons, which can identify multiple intracellular mRNA transcripts. It shows rapid response, high specificity, nuclease stability, and good biocompatibility. Intracellular expts. indicate that the nanoprobe could effectively distinguish cancer cells from their normal cells, even some mRNAs are overexpressed in normal cells. Moreover, it can identify the changes of the expression levels of mRNA in living cells. The current strategy could provide more-accurate information for early cancer detection and availably avoid false pos. results.
- 34Li, N.; Chang, C.; Pan, W.; Tang, B. A Multicolor Nanoprobe for Detection and Imaging of Tumor-Related mRNAs in Living Cells. Angew. Chem., Int. Ed. 2012, 51, 7426– 7430, DOI: 10.1002/anie.201203767Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XpsV2lu78%253D&md5=9a17a709aeebd27cd0f92dc8c421cbd5A multicolor nanoprobe for detection and imaging of tumor-related mRNAs in living cellsLi, Na; Chang, Chenyang; Pan, Wei; Tang, BoAngewandte Chemie, International Edition (2012), 51 (30), 7426-7430, S7426/1-S7426/16CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The authors describe a multicolor fluorescence nanoprobe based on nanoflares, which simultaneously detects three intracellular tumor-related mRNAs. The nanoprobe consists of gold nanoparticles (Au NPs) functionalized with a dense shell of recognition sequences (synthetic oligonucleotides) hybridized to three short dye-terminated reporter sequences by gold-thiol bond formation (Figure I). The gold nanoparticles quench the fluorescence of the dyes. The recognition sequences contain 21-base recognition elements for three specific mRNA transcripts: c-myc mRNA, TK1 mRNA, and GalNAc-T mRNA. In the presence of DNA or RNA targets, the recognition sequences hybridize with the complementary target sequences, forming longer and more stable duplexes, causing the release of the reporter sequences, which can then produce fluorescence signals correlated with the relative amt. of the specific DNA or RNA targets.
- 35Heuer-Jungemann, A.; El-Sagheer, A. H.; Lackie, P. M.; Brown, T.; Kanaras, A. G. Selective Killing of Cells Triggered by Their mRNA Signature in the Presence of Smart Nanoparticles. Nanoscale 2016, 8, 16857– 16861, DOI: 10.1039/C6NR06154KGoogle Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFWlsLbO&md5=afb6f27c37cec1e3011e74a7c176aa87Selective killing of cells triggered by their mRNA signature in the presence of smart nanoparticlesHeuer-Jungemann, Amelie; El-Sagheer, Afaf H.; Lackie, Peter M.; Brown, Tom; Kanaras, Antonios G.Nanoscale (2016), 8 (38), 16857-16861CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)The design of nanoparticles that can selectively perform multiple roles is of utmost importance for the development of the next generation of nanoparticulate drug delivery systems. So far most research studies are focused on the customization of nanoparticulate carriers to maximize their drug loading, enhance their optical signature for tracking in cells or provide photo-responsive effects for therapeutic purposes. However, a vital requirement of the new generation of drug carriers must be the ability to deliver their payload selectively only to cells of interest rather than the majority of various cells in the vicinity. Here we show for the first time a new design of nanoparticulate drug carriers that can specifically distinguish different cell types based on their mRNA signature. These nanoparticles sense and efficiently kill model tumor cells by the delivery of an anti-cancer drug but retain their payload in cells lacking the specific mRNA target.
- 36Lee, J. M.; Dedhar, S.; Kalluri, R.; Thompson, E. W. The Epithelial-Mesenchymal Transition: New Insights in Signaling, Development, and Disease. J. Cell Biol. 2006, 172, 973– 981, DOI: 10.1083/jcb.200601018Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjtF2qsLs%253D&md5=5335036255b1f9c9affcf51ed748129dThe epithelial-mesenchymal transition: New insights in signaling, development, and diseaseLee, Jonathan M.; Dedhar, Shoukat; Kalluri, Raghu; Thompson, Erik W.Journal of Cell Biology (2006), 172 (7), 973-981CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)A review. The conversion of an epithelial cell to a mesenchymal cell is crit. to metazoan embryogenesis and a defining structural feature of organ development. Current interest in this process, which is described as an epithelial-mesenchymal transition (EMT), stems from its developmental importance and its involvement in several adult pathologies. Interest and research in EMT are currently at a high level, as seen by the attendance at the recent EMT meeting in Vancouver, Canada (Oct. 1-3, 2005). The meeting, which was hosted by The EMT International Assocn., was the second international EMT meeting, the first being held in Port Douglas, Queensland, Australia in Oct. 2003. The EMT International Assocn. was formed in 2002 to provide an international body for those interested in EMT and the reverse process, mesenchymal-epithelial transition, and, most importantly, to bring together those working on EMT in development, cancer, fibrosis, and pathol. These themes continued during the recent meeting in Vancouver. Discussion at the Vancouver meeting spanned several areas of research, including signaling pathway activation of EMT and the transcription factors and gene targets involved. Also covered in detail was the basic cell biol. of EMT and its role in cancer and fibrosis, as well as the identification of new markers to facilitate the observation of EMT in vivo. This is particularly important because the potential contribution of EMT during neoplasia is the subject of vigorous scientific debate.
- 37Kang, K. A.; Wang, J.; Jasinski, J. B.; Achilefu, S. Fluorescence Manipulation by Gold Nanoparticles: From Complete Quenching to Extensive Enhancement. J. Nanobiotechnol. 2011, 9, 16, DOI: 10.1186/1477-3155-9-16Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXmtlKkt74%253D&md5=4f85055dd43339debbc0be3c5d24ebceFluorescence manipulation by gold nanoparticles: from complete quenching to extensive enhancementKang, Kyung A.; Wang, Jianting; Jasinski, Jacek B.; Achilefu, SamuelJournal of Nanobiotechnology (2011), 9 (), 16CODEN: JNOAAO; ISSN:1477-3155. (BioMed Central Ltd.)Background: When a fluorophore is placed in the vicinity of a metal nanoparticle possessing a strong plasmon field, its fluorescence emission may change extensively. Our study is to better understand this phenomenon and predict the extent of quenching and/or enhancement of fluorescence, to beneficially utilize it in mol. sensing/imaging. Results: Plasmon field intensities on/around gold nanoparticles (GNPs) with various diams. were theor. computed with respect to the distance from the GNP surface. The field intensity decreased rapidly with the distance from the surface and the rate of decrease was greater for the particle with a smaller diam. Using the plasmon field strength obtained, the level of fluorescence alternation by the field was theor. estd. For exptl. studies, 10 nm GNPs were coated with polymer layer(s) of known thicknesses. Cypate, a near IR fluorophore, was placed on the outermost layer of the polymer coated GNPs, artificially sepd. from the GNP at known distances, and its fluorescence levels were obsd. The fluorescence of Cypate on the particle surface was quenched almost completely and, at approx. 5 nm from the surface, it was enhanced ∼17 times. The level decreased thereafter. Theor. computed fluorescence levels of the Cypate placed at various distances from a 10 nm GNP were compared with the exptl. data. The trend of the resulting fluorescence was similar. The exptl. results, however, showed greater enhancement than the theor. ests., in general. The distance from the GNP surface that showed the max. enhancement in the expt. was greater than the one theor. predicted, probably due to the difference in the two systems. Conclusions: Factors affecting the fluorescence of a fluorophore placed near a GNP are the GNP size, coating material on GNP, wavelengths of the incident light and emitted light and intrinsic quantum yield of the fluorophore. Exptl., we were able to quench and enhance the fluorescence of Cypate, by changing the distance between the fluorophore and GNP. This ability of artificially controlling fluorescence can be beneficially used in developing contrast agents for highly sensitive and specific optical sensing and imaging.
- 38Wu, Z. S.; Jiang, J. H.; Fu, L.; Shen, G. L.; Yu, R. Q. Optical Detection of DNA Hybridization Based on Fluorescence Quenching of Tagged Oligonucleotide Probes by Gold Nanoparticles. Anal. Biochem. 2006, 353, 22– 29, DOI: 10.1016/j.ab.2006.01.018Google ScholarThere is no corresponding record for this reference.
- 39Zheng, D.; Seferos, D. S.; Giljohann, D. A.; Patel, P. C.; Mirkin, C. A. Aptamer Nano-flares for Molecular Detection in Living Cells. Nano Lett. 2009, 9, 3258– 3261, DOI: 10.1021/nl901517bGoogle Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXptlSmtbw%253D&md5=ed0a131b9ccca7002b24570f3032628bAptamer Nano-flares for Molecular Detection in Living CellsZheng, Dan; Seferos, Dwight S.; Giljohann, David A.; Patel, Pinal C.; Mirkin, Chad A.Nano Letters (2009), 9 (9), 3258-3261CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The authors demonstrate a composite nanomaterial, termed an aptamer nano-flare, that can directly quantify an intracellular analyte in a living cell. Aptamer nano-flares consist of a gold nanoparticle core functionalized with a dense monolayer of nucleic acid aptamers with a high affinity for ATP. The probes bind selectively to target mols. and release fluorescent reporters which indicate the presence of the analyte. Addnl., these nanoconjugates are readily taken up by cells where their signal intensity can be used to quantify intracellular analyte concn. These nanoconjugates are a promising approach for the intracellular quantification of other small mols. or proteins, or as agents that use aptamer binding to elicit a biol. response in living systems.
- 40Gilleron, J.; Querbes, W.; Zeigerer, A.; Borodovsky, A.; Marsico, G.; Schubert, U.; Manygoats, K.; Seifert, S.; Andree, C.; Stoeter, M.; Epstein-Barash, H.; Zhang, L.; Koteliansky, V.; Fitzgerald, K.; Fava, E.; Bickle, M.; Kalaidzidis, Y.; Akinc, A.; Maier, M.; Zerial, M. Image-Based Analysis of Lipid Nanoparticle-Mediated siRNA Delivery, Intracellular Trafficking and Endosomal Escape. Nat. Biotechnol. 2013, 31, 638– 646, DOI: 10.1038/nbt.2612Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXpvVymtbg%253D&md5=6fe33f7714c778659e9f87a38808ea9eImage-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escapeGilleron, Jerome; Querbes, William; Zeigerer, Anja; Borodovsky, Anna; Marsico, Giovanni; Schubert, Undine; Manygoats, Kevin; Seifert, Sarah; Andree, Cordula; Stoeter, Martin; Epstein-Barash, Hila; Zhang, Ligang; Koteliansky, Victor; Fitzgerald, Kevin; Fava, Eugenio; Bickle, Marc; Kalaidzidis, Yannis; Akinc, Akin; Maier, Martin; Zerial, MarinoNature Biotechnology (2013), 31 (7), 638-646CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Delivery of short interfering RNAs (siRNAs) remains a key challenge in the development of RNA interference (RNAi) therapeutics. A better understanding of the mechanisms of siRNA cellular uptake, intracellular transport and endosomal release could critically contribute to the improvement of delivery methods. Here we monitored the uptake of lipid nanoparticles (LNPs) loaded with traceable siRNAs in different cell types in vitro and in mouse liver by quant. fluorescence imaging and electron microscopy. We found that LNPs enter cells by both constitutive and inducible pathways in a cell type-specific manner using clathrin-mediated endocytosis as well as macropinocytosis. By directly detecting colloidal-gold particles conjugated to siRNAs, we estd. that escape of siRNAs from endosomes into the cytosol occurs at low efficiency (1-2%) and only during a limited window of time when the LNPs reside in a specific compartment sharing early and late endosomal characteristics. Our results provide insights into LNP-mediated siRNA delivery that can guide development of the next generation of delivery systems for RNAi therapeutics.
- 41Blanco, E.; Shen, H.; Ferrari, M. Principles of Nanoparticle Design for Overcoming Biological Barriers to Drug Delivery. Nat. Biotechnol. 2015, 33, 941– 951, DOI: 10.1038/nbt.3330Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVymt73I&md5=d693b4429d7ad4cc8b3898f7a0fb6235Principles of nanoparticle design for overcoming biological barriers to drug deliveryBlanco, Elvin; Shen, Haifa; Ferrari, MauroNature Biotechnology (2015), 33 (9), 941-951CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Biol. barriers to drug transport prevent successful accumulation of nanotherapeutics specifically at diseased sites, limiting efficacious responses in disease processes ranging from cancer to inflammation. Although substantial research efforts have aimed to incorporate multiple functionalities and moieties within the overall nanoparticle design, many of these strategies fail to adequately address these barriers. Obstacles, such as nonspecific distribution and inadequate accumulation of therapeutics, remain formidable challenges to drug developers. A reimagining of conventional nanoparticles is needed to successfully negotiate these impediments to drug delivery. Site-specific delivery of therapeutics will remain a distant reality unless nanocarrier design takes into account the majority, if not all, of the biol. barriers that a particle encounters upon i.v. administration. By successively addressing each of these barriers, innovative design features can be rationally incorporated that will create a new generation of nanotherapeutics, realizing a paradigmatic shift in nanoparticle-based drug delivery.
- 42Perez-Arnaiz, C.; Busto, N.; Leal, J. M.; Garcia, B. New Insights into the Mechanism of the DNA/Doxorubicin Interaction. J. Phys. Chem. B 2014, 118, 1288– 1295, DOI: 10.1021/jp411429gGoogle Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXnsV2qtQ%253D%253D&md5=65a09adebe62d60071b731847caed1a1New Insights into the Mechanism of the DNA/Doxorubicin InteractionPerez-Arnaiz, Cristina; Busto, Natalia; Leal, Jose M.; Garcia, BegonaJournal of Physical Chemistry B (2014), 118 (5), 1288-1295CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)Doxorubicin (DOX) is an important anthracycline antibiotic whose intricate features of binding to DNAs, not yet fully understood, have been the object of intense debate. The dimerization equil. has been studied at pH = 7.0, I = 2.5 mM, and T = 25°. A thermodn. and kinetic study of the binding of doxorubicin to DNA, carried out by CD, viscometry, differential scanning calorimetry, fluorescence, isothermal titrn. calorimetry, and T-jump relaxation measurements, has enabled us to characterize for the first time two different types of calf thymus DNA (ctDNA)/DOX complexes: PD1 for CDOX/CDNA < 0.3, and PD2 for higher drug content. The nature of the PD1 complex is described better in light of the affinity of DOX with the synthetic copolymers [poly(dA-dT)]2 and [poly(dG-dC)]2. The formation of PD1 has been categorized kinetically as a two-step mechanism in which the fast step is the groove binding in the AT region, and the slow step is the intercalation into the GC region. This bifunctional nature provides a plausible explanation for the high PD1 const. obtained (K1 = 2.3 × 108 M-1). Moreover, the formation of an external aggregate complex ctDNA/DOX (PD2) at the expense of PD1, with K2 = 9.3 × 105 M-1, has been evinced.
- 43Hajihassan, Z.; Rabbani-Chadegani, A. Studies on the Binding Affinity of Anticancer Drug Mitoxantrone to Chromatin, DNA and Histone Proteins. J. Biomed. Sci. 2009, 16, 31, DOI: 10.1186/1423-0127-16-31Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1M3jtVanuw%253D%253D&md5=9dd75cc4aaccdbf234b8d5071cc4606cStudies on the binding affinity of anticancer drug mitoxantrone to chromatin, DNA and histone proteinsHajihassan Zahra; Rabbani-Chadegani AzraJournal of biomedical science (2009), 16 (), 31 ISSN:.Mitoxantrone is a potent antitumor drug, widely used in the treatment of various cancers. In the present study, we have investigated and compared the affinity of anticancer drug, mitoxantrone, to EDTA-soluble chromatin (SE-chromatin), DNA and histones employing UV/Vis, fluorescence, CD spectroscopy, gel electrophoresis and equilibrium dialysis techniques. The results showed that the interaction of mitoxantrone with SE-chromatin proceeds into compaction/aggregation as revealed by reduction in the absorbencies at 608 and 260 nm (hypochromicity) and disappearance of both histones and DNA on the gels. Mitoxantrone interacts strongly with histone proteins in solution making structural changes in the molecule as shown by CD and fluorescence analysis. The binding isotherms demonstrate a positive cooperative binding pattern for the chromatin- mitoxantrone interaction. It is suggested higher binding affinity of mitoxantrone to chromatin compared to DNA implying that the histone proteins may play an important role in the chromatin- mitoxantrone interaction process.
- 44Ohnuma, T.; Matsumoto, T.; Itoi, A.; Kawana, A.; Nishiyama, T.; Ogura, K.; Hiratsuka, A. Enhanced Sensitivity of A549 Cells to the Cytotoxic Action of Anticancer Drugs via Suppression of Nrf2 by Procyanidins from Cinnamomi Cortex Extract. Biochem. Biophys. Res. Commun. 2011, 413, 623– 629, DOI: 10.1016/j.bbrc.2011.09.014Google ScholarThere is no corresponding record for this reference.
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Abstract
Scheme 1
Scheme 1. Schematic Illustration of the Multiplexed Nanoparticle Dimer and the Process of mRNA DetectionaaWhen 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 (Dye 3).
Figure 1
Figure 1. Confocal microscopy images of cells incubated with (A–C) nanoparticle dimers and (D–F) “non-targeting” scramble nanoparticle dimers. Sixteen HBE cells express only keratin 8, and MRC 5 cells express only vimentin. A549 expresses both keratin 8 and vimentin. A fluorescence signal corresponding to the presence of: keratin 8 mRNA (A), vimentin mRNA (B), and both vimentin and keratin 8 mRNA (C) is observed. All three cell lines display no response when incubated with nanoparticle dimers designed with “non-targeting” scramble sequences. Color guide: Keratin 8 “flare” strand, green; vimentin flare strand, red; vimentin and keratin 8 sense strand, blue; scrambled flare strand, green; scrambled sense strand, blue; nuclear counterstain, white. Scale bars are 15 μm.
Scheme 2
Scheme 2. Schematic Illustration of Nanoparticle Dimer Used in Drug Release Experimentsaa(Left) Dimer loaded with DOX (Drug 1) and MTX (Drug 2). No fluorophores were used in flare and sense strands. Both DOX and MTX have fluorescent properties, which are quenched due to the close proximity to the AuNP core. When the target mRNA binds to the sense strand, the intercalated drugs are released (right) causing an increase in the fluorescence signal.
Figure 2
Figure 2. Confocal microscopy images of live cells incubated with nanoparticle dimers. The nanoparticle dimers were loaded with DOX and MXT (left column). The 16HBE cells show release of only DOX, corresponding to the presence of keratin 8 mRNA (see Figure 1). In MRC 5 cells, MTX was released corresponding to the presence of vimentin mRNA. In A549 cells, both keratin 8 and vimentin mRNA were detected, therefore both drugs MTX and DOX were released. Scramble nanoparticle dimers (D–F) did not display any fluorescence signal indicating that they retained their drug payload. Color guide: DOX release, green; MXT release, red; nuclear counterstain, white. Scale bar is 15 μm.
Figure 3
Figure 3. Trypan blue cell viability assay of (A) 16 HBE (B) MRC 5 and (C) A 549 cells incubated with various nanoparticle designs. Cells were incubated with: (2) nanoparticles sensing all mRNAs (gmRNA); (3) nanoparticles functionalized with scramble “non-targeting” oligonucleotides loaded with DOX and MXT; (4) free DOX; (5) free MTX; (6) nanoparticle dimers designed to sense keratin8 and vimentin mRNA loaded with DOX and MTX. Data are shown as mean ± SEM (n = 3). P-values were determined by a one-way ANOVA. *p < 0.05, ****p < 0.0001.
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- 2Shenashen, M. A.; El-Safty, S. A.; Elshehy, E. A. Synthesis, Morphological Control, and Properties of Silver Nanoparticles in Potential Applications. Part. Part. Syst. Char. 2014, 31, 293– 316, DOI: 10.1002/ppsc.2013001812https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXkt1Sju7c%253D&md5=e1a3e3cb95619f5ab0499e1dbd0f5d77Synthesis, Morphological Control, and Properties of Silver Nanoparticles in Potential ApplicationsShenashen, Mohamed A.; El-Safty, Sherif A.; Elshehy, Emad A.Particle & Particle Systems Characterization (2014), 31 (3), 293-316CODEN: PPCHEZ; ISSN:1521-4117. (Wiley-VCH Verlag GmbH & Co. KGaA)Nanostructured materials, esp. nanoparticles (NPs), of noble metal NPs such as silver (Ag) have been the focus of research in recent decades because of their distinct phys., chem., and biol. properties. These materials have attracted considerable attention because of their potential applications, such as catalysis, biosensing, drug delivery, and nanodevice fabrication. Previous studies on Ag NPs have clearly demonstrated that their electromagnetic, optical, and catalytic properties are strongly influenced by their shape, size, and size distribution, which can be varied by using different synthetic methods, reducing agents, and stabilizers. The valuable optical properties of Ag NPs have allowed for new approaches in sensing and imaging applications, offering a wide range of detection modes, such as colorimetric, scattering, and surface-enhanced Raman scattering techniques, at extremely low detection limits. Here, an overview of the various chem., phys., and biol. properties of Ag NP fabrication approaches to obtain the various shapes and sizes is presented.
- 3Pankhurst, Q.; Jones, S.; Dobson, J. Applications of Magnetic Nanoparticles in Biomedicine: The Story so Far. J. Phys. D: Appl. Phys. 2016, 49, 501002, DOI: 10.1088/0022-3727/49/50/5010023https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsFSru7fJ&md5=51a321039788a5ca12cb95d61af9bb99Applications of magnetic nanoparticles in biomedicine: the story so farPankhurst, Quentin; Jones, Stephen; Dobson, JonJournal of Physics D: Applied Physics (2016), 49 (50), 501002/1-501002/2CODEN: JPAPBE; ISSN:0022-3727. (IOP Publishing Ltd.)There is no expanded citation for this reference.
- 4Ling, D.; Lee, N.; Hyeon, T. Chemical Synthesis and Assembly of Uniformly Sized Iron Oxide Nanoparticles for Medical Applications. Acc. Chem. Res. 2015, 48, 1276– 1285, DOI: 10.1021/acs.accounts.5b000384https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXntlCmtb4%253D&md5=5623cdbf613bc8c00ae0725ec6f2b316Chemical Synthesis and Assembly of Uniformly Sized Iron Oxide Nanoparticles for Medical ApplicationsLing, Daishun; Lee, Nohyun; Hyeon, TaeghwanAccounts of Chemical Research (2015), 48 (5), 1276-1285CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. Magnetic iron oxide nanoparticles have been extensively investigated for their various biomedical applications including diagnostic imaging, biol. sensing, drug, cell, and gene delivery, and cell tracking. Recent advances in the designed synthesis and assembly of uniformly sized iron oxide nanoparticles have brought innovation in the field of nanomedicine. This Account provides a review on the recent progresses in the controlled synthesis and assembly of uniformly sized iron oxide nanoparticles for medical applications. In particular, it focuses on three topics: stringent control of particle size during synthesis via the "heat-up" process, surface modification for the high stability and biocompatibility of the nanoparticles for diagnostic purposes, and assembly of the nanoparticles within polymers or mesoporous silica matrixes for theranostic applications. Using extremely small 3 nm sized iron oxide nanoparticles (ESION), a new nontoxic T1 MRI contrast agent was realized for high-resoln. MRI of blood vessels down to 0.2 mm. Ferrimagnetic iron oxide nanoparticles (FION) that are larger than 20 nm exhibit extremely large magnetization and coercivity values. The cells labeled with FIONs showed very high T2 contrast effect so that even a single cell can be readily imaged. Designed assembly of iron oxide nanoparticles with mesoporous silica and polymers was conducted to fabricate multifunctional nanoparticles for theranostic applications. Mesoporous silica nanoparticles are excellent scaffolds for iron oxide nanoparticles, providing magnetic resonance and fluorescence imaging modalities as well as the functionality of the drug delivery vehicle. Polymeric ligands could be designed to respond to various biol. stimuli such as pH, temp., and enzymic activity. For example, we fabricated tumor pH-sensitive magnetic nanogrenades (termed PMNs) composed of self-assembled iron oxide nanoparticles and pH-responsive ligands. They were utilized to visualize small tumors (<3 mm) via pH-responsive T1 MRI and fluorescence imaging. Also, superior photodynamic therapeutic efficacy in highly drug-resistant heterogeneous tumors was obsd. We expect that these multifunctional and bioresponsive nanoplatforms based on uniformly sized iron oxide nanoparticles will provide more unique theranostic approaches in clin. uses.
- 5Giljohann, D. A.; Seferos, D. S.; Daniel, W. L.; Massich, M. D.; Patel, P. C.; Mirkin, C. A. Gold Nanoparticles for Biology and Medicine. Angew. Chem., Int. Ed. 2010, 49, 3280– 3294, DOI: 10.1002/anie.2009043595https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXlt1GlsbY%253D&md5=0809d2a7d09124a8975d5ea77e679442Gold Nanoparticles for Biology and MedicineGiljohann, David A.; Seferos, Dwight S.; Daniel, Weston L.; Massich, Matthew D.; Patel, Pinal C.; Mirkin, Chad A.Angewandte Chemie, International Edition (2010), 49 (19), 3280-3294CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Gold colloids have fascinated scientists for over a century and are now heavily utilized in chem., biol., engineering, and medicine. Today these materials can be synthesized reproducibly, modified with seemingly limitless chem. functional groups, and, in certain cases, characterized with at.-level precision. This Review highlights recent advances in the synthesis, bioconjugation, and cellular uses of gold nanoconjugates. There are now many examples of highly sensitive and selective assays based upon gold nanoconjugates. In recent years, focus has turned to therapeutic possibilities for such materials. Structures which behave as gene-regulating agents, drug carriers, imaging agents, and photoresponsive therapeutics have been developed and studied in the context of cells and many debilitating diseases. These structures are not simply chosen as alternatives to mol.-based systems, but rather for their new phys. and chem. properties, which confer substantive advantages in cellular and medical applications.
- 6Alivisatos, A. P.; Johnsson, K. P.; Peng, X. G.; Wilson, T. E.; Loweth, C. J.; Bruchez, M. P.; Schultz, P. G. Organization of ’Nanocrystal Molecules’ Using DNA. Nature 1996, 382, 609– 611, DOI: 10.1038/382609a06https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XltVWqsrY%253D&md5=27e1eb981a0cfd51907eff5a5a105f50Organization of 'nanocrystal molecules' using DNAAlivisatos, A. Paul; Johnsson, Kai P.; Peng, Xiaogang; Wilson, Troy E.; Loweth, Colin J.; Bruchez, Marcel P., Jr.; Schultz, Peter G.Nature (London) (1996), 382 (6592), 609-611CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)The authors describe a strategy for the synthesis of 'nanocrystal mols.', in which discrete nos. of Au nanocrystals are organized into spatially defined structures based on Watson-Crick base-pairing interactions. The authors attach single-stranded DNA oligonucleotides of defined length and sequence to individual nanocrystals, and these assemble into dimers and trimers on addn. of a complementary single-stranded DNA template. The authors anticipate that this approach should allow the construction of more complex two- and three-dimensional assemblies.
- 7Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. A DNA-Based Method for Rationally Assembling Nanoparticles Into Macroscopic Materials. Nature 1996, 382, 607– 609, DOI: 10.1038/382607a07https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XltVWqsrk%253D&md5=11128671758a23f01bf6c8d3b20b3921A DNA-based method for rationally assembling nanoparticles into macroscopic materialsMirkin, Chad A.; Letsinger, Robert L.; Mucic, Robert C.; Storhoff, James J.Nature (London) (1996), 382 (6592), 607-609CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)Colloidal particles of metals and semiconductors have potentially useful optical, optoelectronic and material properties that derive from their small (nanoscopic) size. These properties might lead to applications including chem. sensors, spectroscopic enhancers, quantum dot and nanostructure fabrication, and microimaging methods. A great deal of control can now be exercised over the chem. compn., size and polydispersity of colloidal particles, and many methods have been developed for assembling them into useful aggregates and materials. Here we describe a method for assembling colloidal gold nanoparticles rationally and reversibly into macroscopic aggregates. The method involves attaching to the surfaces of two batches of 13-nm gold particles non-complementary DNA oligonucleotides capped with thiol groups, which bind to gold. When we add to the soln. an oligonucleotide duplex with 'sticky ends' that are complementary to the two grafted sequences, the nanoparticles self-assemble into aggregates. This assembly process can be reversed by thermal denaturation. This strategy should now make it possible to tailor the optical, electronic and structural properties of the colloidal aggregates by using the specificity of DNA interactions to direct the interactions between particles of different size and compn.
- 8Cutler, J. I.; Auyeung, E.; Mirkin, C. A. Spherical Nucleic Acids. J. Am. Chem. Soc. 2012, 134, 1376– 1391, DOI: 10.1021/ja209351u8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XktVKlsQ%253D%253D&md5=0ccb9d7fab59aee9e0984c4e7d905d37Spherical Nucleic AcidsCutler, Joshua I.; Auyeung, Evelyn; Mirkin, Chad A.Journal of the American Chemical Society (2012), 134 (3), 1376-1391CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A review. A historical perspective of the development of spherical nucleic acid (SNA) conjugates and other three-dimensional nucleic acid nanostructures is provided. This Perspective details the synthetic methods for prepg. them, followed by a discussion of their unique properties and theor. and exptl. models for understanding them. Important examples of technol. advances made possible by their fundamental properties spanning the fields of chem., mol. diagnostics, gene regulation, medicine, and materials science are also presented.
- 9Hurst, S. J.; Hill, H. D.; Mirkin, C. A. ″Three-Dimensional Hybridization″ With Polyvalent DNA-Gold Nanoparticle Conjugates. J. Am. Chem. Soc. 2008, 130, 12192– 12200, DOI: 10.1021/ja804266jThere is no corresponding record for this reference.
- 10Jones, M. R.; Macfarlane, R. J.; Lee, B.; Zhang, J. A.; Young, K. L.; Senesi, A. J.; Mirkin, C. A. DNA-Nanoparticle Superlattices Formed From Anisotropic Building Blocks. Nat. Mater. 2010, 9, 913– 917, DOI: 10.1038/nmat287010https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtlWkt7zK&md5=6e6c918813e2bd715c9a3c44203d38ddDNA-nanoparticle superlattices formed from anisotropic building blocksJones, Matthew R.; MacFarlane, Robert J.; Lee, Byeongdu; Zhang, Jian; Young, Kaylie L.; Senesi, Andrew J.; Mirkin, Chad A.Nature Materials (2010), 9 (11), 913-917CODEN: NMAACR; ISSN:1476-1122. (Nature Publishing Group)Directional bonding interactions in solid-state at. lattices dictate the unique symmetries of at. crystals, resulting in a diverse and complex assortment of three-dimensional structures that exhibit a wide variety of material properties. Methods to create analogous nanoparticle superlattices are beginning to be realized, but the concept of anisotropy is still largely underdeveloped in most particle assembly schemes. Some examples provide interesting methods to take advantage of anisotropic effects, but most are able to make only small clusters or lattices that are limited in crystallinity and esp. in lattice parameter programmability. Anisotropic nanoparticles can be used to impart directional bonding interactions on the nanoscale, both through face-selective functionalization of the particle with recognition elements to introduce the concept of valency, and through anisotropic interactions resulting from particle shape. The authors examine the concept of inherent shape-directed crystn. in the context of DNA-mediated nanoparticle assembly. Importantly, the anisotropy of these particles can be used to synthesize 1-, two- and three-dimensional structures that cannot be made through the assembly of spherical particles.
- 11Park, S. Y.; Lytton-Jean, A. K. R.; Lee, B.; Weigand, S.; Schatz, G. C.; Mirkin, C. A. DNA-Programmable Nanoparticle Crystallization. Nature 2008, 451, 553– 556, DOI: 10.1038/nature0650811https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhs1ent7g%253D&md5=b5c8c1ab8b7f43d32793560e4869ad2fDNA-programmable nanoparticle crystallizationPark, Sung Yong; Lytton-Jean, Abigail K. R.; Lee, Byeongdu; Weigand, Steven; Schatz, George C.; Mirkin, Chad A.Nature (London, United Kingdom) (2008), 451 (7178), 553-556CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)It was first shown more than ten years ago that DNA oligonucleotides can be attached to gold nanoparticles rationally to direct the formation of larger assemblies. Since then, oligonucleotide-functionalized nanoparticles have been developed into powerful diagnostic tools for nucleic acids and proteins, and into intracellular probess and gene regulators. In contrast, the conceptually simple yet powerful idea that functionalized nanoparticles might serve as basic building blocks that can be rationally assembled through programmable base-pairing interactions into highly ordered macroscopic materials remains poorly developed. So far, the approach has mainly resulted in polymn., with modest control over the placement of, the periodicity in, and the distance between particles within the assembled material. That is, most of the materials obtained thus far are best classified as amorphous polymers, although a few examples of colloidal crystal formation exist. Here, we demonstrate that DNA can be used to control the crystn. of nanoparticle-oligonucleotide conjugates to the extent that different DNA sequences guide the assembly of the same type of inorg. nanoparticle into different cryst. states. We show that the choice of DNA sequences attached to the nanoparticle building blocks, the DNA linking mols. and the absence or presence of a non-bonding single-base flexor can be adjusted so that gold nanoparticles assemble into micrometer-sized face-centered-cubic or body-centered-cubic crystal structures. Our findings thus clearly demonstrate that synthetically programmable colloidal crystn. is possible, and that a single-component system can be directed to form different structures.
- 12Park, S. J.; Lazarides, A. A.; Storhoff, J. J.; Pesce, L.; Mirkin, C. A. The Structural Characterization of Oligonucleotide-Modified Gold Nanoparticle Networks Formed by DNA Hybridization. J. Phys. Chem. B 2004, 108, 12375– 12380, DOI: 10.1021/jp040242b12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXlvFGisLw%253D&md5=7c40303add3bf2ba27d47b79e4f9fda6The Structural Characterization of Oligonucleotide-Modified Gold Nanoparticle Networks Formed by DNA HybridizationPark, So-Jung; Lazarides, Anne A.; Storhoff, James J.; Pesce, Lorenzo; Mirkin, Chad A.Journal of Physical Chemistry B (2004), 108 (33), 12375-12380CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)The structural properties of DNA-linked gold nanoparticle materials were examd. using synchrotron small-angle X-ray scattering (SAXS). The materials are composed of 12 or 19 nm diam. gold particles modified with 3' or 5' alkylthiol-capped 12-base oligonucleotides and linked with complementary oligonucleotides. Structure factors were derived from scattering intensities, and nearest-neighbor distances were detd. from the primary peak in the pair distance distribution functions. The sepn. between particles was found to increase linearly with DNA linker length for 24, 48, and 72 base pair linkers. For assemblies formed in 0.3 M NaCl, 10 mM phosphate buffer soln., the increment in the interparticle distance was found to be 2.5 Å per base pair. Particle sepns. in assemblies at lower electrolyte concn. were larger, indicating that dielec. screening modulates the interactions. The effect of DNA sequence was studied with poly-adenine or poly-thymine spacer sequences incorporated between the alkylthiol and recognition sequences. The assemblies with poly-adenine spacer sequences showed significantly shorter particle sepns. than the assemblies involving poly-thymine spacers, a consequence of their different affinities for the gold surface. While the scattering data do not display evidence of long-range order, pair distance distribution functions indicate the presence of short-range order.
- 13Nykypanchuk, D.; Maye, M. M.; van der Lelie, D.; Gang, O. DNA-Guided Crystallization of Colloidal Nanoparticles. Nature 2008, 451, 549– 552, DOI: 10.1038/nature0656013https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhs1ent7s%253D&md5=dba644c8f297d79a3a9cc5d1b64ad9c3DNA-guided crystallization of colloidal nanoparticlesNykypanchuk, Dmytro; Maye, Matthew M.; van der Lelie, Daniel; Gang, OlegNature (London, United Kingdom) (2008), 451 (7178), 549-552CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Many nanometer-size building blocks will readily assemble into macroscopic structures. If the process is accompanied by effective control over the interactions between the blocks and all entropic effects, then the resultant structures will be ordered with a precision hard to achieve with other fabrication methods. But it remains challenging to use self-assembly to design systems comprised of different types of building blocks - to realize novel magnetic, plasmonic and photonic metamaterials, for example. A conceptually simple idea for overcoming this problem is the use of 'encodable' interactions between building blocks; this can in principle be straightforwardly implemented using biomols. Strategies that use DNA programmability to control the placement of nanoparticles in one and two dimensions have indeed been demonstrated. However, theor. understanding of how to extend this approach to 3 dimensions is limited, and most expts. have yielded amorphous aggregates and only occasionally crystallites of close-packed micrometer-size particles. Here, the authors report the formation of 3-dimensional cryst. assemblies of gold nanoparticles mediated by interactions between complementary DNA mols. attached to the nanoparticles' surface. We find that the nanoparticle crystals form reversibly during heating and cooling cycles. Moreover, the body-centered-cubic lattice structure is temp.-tuneable and structurally open, with particles occupying only ∼4% of the unit cell vol. The authors expect that the DNA-mediated crystn. approach, and the insight into DNA design requirements it has provided, will facilitate both the creation of new classes of ordered multicomponent metamaterials and the exploration of the phase behavior of hybrid systems with addressable interactions.
- 14Harimech, P. K.; Gerrard, S. R.; El-Sagheer, A. H.; Brown, T.; Kanaras, A. G. Reversible Ligation of Programmed DNA-Gold Nanoparticle Assemblies. J. Am. Chem. Soc. 2015, 137, 9242– 9245, DOI: 10.1021/jacs.5b0568314https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtF2ltr7J&md5=5f440a69ae6c73433d873ceeadbf4becReversible Ligation of Programmed DNA-Gold Nanoparticle AssembliesHarimech, Pascal K.; Gerrard, Simon R.; El-Sagheer, Afaf H.; Brown, Tom; Kanaras, Antonios G.Journal of the American Chemical Society (2015), 137 (29), 9242-9245CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We demonstrate a new method to reversibly crosslink DNA-nanoparticle dimers, trimers, and tetramers using light as an external stimulus. A DNA interstrand photo-crosslinking reaction is possible via ligation of a cyano-vinyl carbazole nucleoside with an opposite thymine when irradiated at 365 nm. This reaction results in nanoparticle assemblies that are not susceptible to DNA dehybridization conditions. The chem. bond between the two complementary DNA strands can be reversibly broken upon light irradn. at 312 nm. This is the first example of reversible ligation in DNA-nanoparticle assemblies using light and enables new developments in the field of programmed nanoparticle organization.
- 15Heuer-Jungemann, A.; Kirkwood, R.; El-Sagheer, A. H.; Brown, T.; Kanaras, A. G. Copper-Free Click Chemistry as an Emerging Tool for the Programmed Ligation of DNA-Functionalised Gold Nanoparticles. Nanoscale 2013, 5, 7209– 7212, DOI: 10.1039/c3nr02362a15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtFKgu7vI&md5=fc12ba5108d6bb99b25ee7f6efaed8d1Copper-free click chemistry as an emerging tool for the programmed ligation of DNA-functionalised gold nanoparticlesHeuer-Jungemann, Amelie; Kirkwood, Robert; El-Sagheer, Afaf H.; Brown, Tom; Kanaras, Antonios G.Nanoscale (2013), 5 (16), 7209-7212CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)We demonstrate a new method to program the ligation of single stranded DNA-modified gold nanoparticles using copper-free click chem. Gold nanoparticles functionalized with a discrete no. of 3'-azide or 5'-alkyne modified oligonucleotides, can be brought together via a splint strand and covalently 'clicked', in a simple one-pot reaction. This new approach to the assembly of gold nanoparticles is inherently advantageous in comparison to the traditional enzymic ligation. The chem. ligation is specific and takes place at room temp. by simply mixing the particles without the need for special enzymic conditions. The yield of 'clicked' nanoparticles can be as high as 92%. The ease of the copper-free, click-ligation' method allows for its universal applicability and opens up new avenues in programmed nanoparticle organization.
- 16Hurst, S. J.; Lytton-Jean, A. K. R.; Mirkin, C. A. Maximizing DNA Loading on a Range of Gold Nanoparticle Sizes. Anal. Chem. 2006, 78, 8313– 8318, DOI: 10.1021/ac061358216https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFyhtrfO&md5=1b1f5ac1386517068d1252b850112211Maximizing DNA Loading on a Range of Gold Nanoparticle SizesHurst, Sarah J.; Lytton-Jean, Abigail K. R.; Mirkin, Chad A.Analytical Chemistry (2006), 78 (24), 8313-8318CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)The authors have investigated the variables that influence DNA coverage on gold nanoparticles. The effects of salt concn., spacer compn., nanoparticle size, and degree of sonication have been evaluated. Maximum loading was obtained by salt aging the nanoparticles to ∼0.7 M NaCl in the presence of DNA contg. a poly(ethylene glycol) spacer. In addn., DNA loading was substantially increased by sonicating the nanoparticles during the surface loading process. Last, nanoparticles up to 250 nm in diam. were found have ∼2 orders of magnitude higher DNA loading than smaller (13-30 nm) nanoparticles, a consequence of their larger surface area. Stable large particles are attractive for a variety of biodiagnostic assays.
- 17Choi, C. H. J.; Hao, L.; Narayan, S. P.; Auyeung, E.; Mirkin, C. A. Mechanism for the Endocytosis of Spherical Nucleic Acid Nanoparticle Conjugates. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 7625– 7630, DOI: 10.1073/pnas.130580411017https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXptFGrt7k%253D&md5=f0755c5fa2b33b5ca167ef14c6394b27Mechanism for the endocytosis of spherical nucleic acid nanoparticle conjugatesChoi, Chung Hang J.; Hao, Liangliang; Narayan, Suguna P.; Auyeung, Evelyn; Mirkin, Chad A.Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (19), 7625-7630, S7625/1-S7625/7CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Intracellular delivery of nucleic acids as gene regulation agents typically requires the use of cationic carriers or viral vectors, yet issues related to cellular toxicity or immune responses hamper their attractiveness as therapeutic candidates. The discovery that spherical nucleic acids (SNAs), polyanionic structures comprised of densely packed, highly oriented oligonucleotides covalently attached to the surface of nanoparticles, can effectively enter more than 50 different cell types presents a potential strategy for overcoming the limitations of conventional transfection agents. Unfortunately, little is known about the mechanism of endocytosis of SNAs, including the pathway of entry and specific proteins involved. Here, we demonstrate that the rapid cellular uptake kinetics and intracellular transport of SNAs stem from the arrangement of oligonucleotides into a 3D architecture, which supports their targeting of class A scavenger receptors and endocytosis via a lipid-raft-dependent, caveolae-mediated pathway. These results reinforce the notion that SNAs can serve as therapeutic payloads and targeting structures to engage biol. pathways not readily accessible with linear oligonucleotides.
- 18Wu, X. A.; Choi, C. H. J.; Zhang, C.; Hao, L.; Mirkin, C. A. Intracellular Fate of Spherical Nucleic Acid Nanoparticle Conjugates. J. Am. Chem. Soc. 2014, 136, 7726– 7733, DOI: 10.1021/ja503010a18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXotFOhs7k%253D&md5=b2e703b091edc7eb39931e96ba5c4b68Intracellular Fate of Spherical Nucleic Acid Nanoparticle ConjugatesWu, Xiaochen A.; Choi, Chung Hang J.; Zhang, Chuan; Hao, Liangliang; Mirkin, Chad A.Journal of the American Chemical Society (2014), 136 (21), 7726-7733CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Spherical nucleic acid (SNA) nanoparticle conjugates are a class of bionanomaterials that are extremely potent in many biomedical applications. Their unique ability to enter multiple mammalian cell types as single-entity agents arises from their novel three-dimensional architecture, which consists of a dense shell of highly oriented oligonucleotides chem. attached typically to a gold nanoparticle core. This architecture allows SNAs to engage certain cell surface receptors to facilitate entry. Here, we report studies aimed at detg. the intracellular fate of SNAs and the trafficking events that occur inside C166 mouse endothelial cells after cellular entry. We show that SNAs traffic through the endocytic pathway into late endosomes and reside there for up to 24 h after incubation. Disassembly of oligonucleotides from the nanoparticle core is obsd. 16 h after cellular entry, most likely due to degrdn. by enzymes such as DNase II localized in late endosomes. Our observations point to these events being likely independent of core compn. and treatment conditions, and they do not seem to be particularly dependent upon oligonucleotide sequence. Significantly and surprisingly, the SNAs do not enter the lysosomes under the conditions studied. To independently track the fate of the particle core and the fluorophore-labeled oligonucleotides that comprise its shell, we synthesized a novel class of quantum dot SNAs to det. that as the SNA structures are broken down over the 24 h time course of the expt., the oligonucleotide fragments are recycled out of the cell while the nanoparticle core is not. This mechanistic insight points to the importance of designing and synthesizing next-generation SNAs that can bypass the degrdn. bottleneck imposed by their residency in late endosomes, and it also suggests that such structures might be extremely useful for endosomal signaling pathways by engaging receptors that are localized within the endosome.
- 19Giljohann, D. A.; Seferos, D. S.; Patel, P. C.; Millstone, J. E.; Rosi, N. L.; Mirkin, C. A. Oligonucleotide Loading Determines Cellular Uptake of DNA-Modified Gold Nanoparticles. Nano Lett. 2007, 7, 3818– 3821, DOI: 10.1021/nl072471q19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht1yrurvM&md5=46417b236e7d7075a484007a474f9acbOligonucleotide Loading Determines Cellular Uptake of DNA-Modified Gold NanoparticlesGiljohann, David A.; Seferos, Dwight S.; Patel, Pinal C.; Millstone, Jill E.; Rosi, Nathaniel L.; Mirkin, Chad A.Nano Letters (2007), 7 (12), 3818-3821CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The cellular internalization of oligonucleotide-modified nanoparticles is investigated. Uptake is dependent on the d. of the oligonucleotide loading on the surface of the particles, where higher densities lead to greater uptake. Densely functionalized nanoparticles adsorb a large no. of proteins on the nanoparticle surface. Nanoparticle uptake is greatest where a large no. of proteins are assocd. with the particle.
- 20Patel, P. C.; Giljohann, D. A.; Daniel, W. L.; Zheng, D.; Prigodich, A. E.; Mirkin, C. A. Scavenger Receptors Mediate Cellular Uptake of Polyvalent Oligonucleotide-Functionalized Gold Nanoparticles. Bioconjugate Chem. 2010, 21, 2250– 2256, DOI: 10.1021/bc100242320https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVSisbbF&md5=d907b2d964481128d4888696ef1dc22cScavenger Receptors Mediate Cellular Uptake of Polyvalent Oligonucleotide-Functionalized Gold NanoparticlesPatel, Pinal C.; Giljohann, David A.; Daniel, Weston L.; Zheng, Dan; Prigodich, Andrew E.; Mirkin, Chad A.Bioconjugate Chemistry (2010), 21 (12), 2250-2256CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)Mammalian cells have been shown to internalize oligonucleotide-functionalized gold nanoparticles (DNA-Au NPs or siRNA-Au NPs) without the aid of auxiliary transfection agents and use them to initiate an antisense or RNAi response. Previous studies have shown that the dense monolayer of oligonucleotides on the nanoparticle leads to the adsorption of serum proteins and facilitates cellular uptake. Here, we show that serum proteins generally act to inhibit cellular uptake of DNA-Au NPs. We identify the pathway for DNA-Au NP entry in HeLa cells. Biochem. analyses indicate that DNA-Au NPs are taken up by a process involving receptor-mediated endocytosis. Evidence shows that DNA-Au NP entry is primarily mediated by scavenger receptors, a class of pattern-recognition receptors. This uptake mechanism appears to be conserved across species, as blocking the same receptors in mouse cells also disrupted DNA-Au NP entry. Polyvalent nanoparticles functionalized with siRNA are shown to enter through the same pathway. Thus, scavenger receptors are required for cellular uptake of polyvalent oligonucleotide functionalized nanoparticles.
- 21Seferos, D. S.; Prigodich, A. E.; Giljohann, D. A.; Patel, P. C.; Mirkin, C. A. Polyvalent DNA Nanoparticle Conjugates Stabilize Nucleic Acids. Nano Lett. 2009, 9, 308– 311, DOI: 10.1021/nl802958f21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsFantbfL&md5=9c13243d65bae4af040f17b8d828444fPolyvalent DNA Nanoparticle Conjugates Stabilize Nucleic AcidsSeferos, Dwight S.; Prigodich, Andrew E.; Giljohann, David A.; Patel, Pinal C.; Mirkin, Chad A.Nano Letters (2009), 9 (1), 308-311CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Polyvalent oligonucleotide gold nanoparticle conjugates have unique fundamental properties including distance-dependent plasmon coupling, enhanced binding affinity, and the ability to enter cells and resist enzymic degrdn. Stability in the presence of enzymes is a key consideration for therapeutic uses; however the manner and mechanism by which such nanoparticles are able to resist enzymic degrdn. is unknown. Here, we quantify the enhanced stability of polyvalent gold oligonucleotide nanoparticle conjugates with respect to enzyme-catalyzed hydrolysis of DNA and present evidence that the neg. charged surfaces of the nanoparticles and resultant high local salt concns. are responsible for enhanced stability.
- 22Heuer-Jungemann, A.; Harimech, P. K.; Brown, T.; Kanaras, A. G. Gold Nanoparticles and Fluorescently-Labelled DNA as a Platform for Biological Sensing. Nanoscale 2013, 5, 9503– 9510, DOI: 10.1039/c3nr03707j22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsFaltrbP&md5=1cfc1c9836f3c35d00cf14180c621116Gold nanoparticles and fluorescently-labelled DNA as a platform for biological sensingHeuer-Jungemann, Amelie; Harimech, Pascal K.; Brown, Tom; Kanaras, Antonios G.Nanoscale (2013), 5 (20), 9503-9510CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)A review. In the past decade gold nanoparticle-nucleic acid conjugates became progressively important for biomedical applications. Fluorophores attached to nucleic acid-gold nanoparticle conjugates have opened up a new era of biol. sensing. The most promising advancement in this field was the invention of the so-called nano-flare' systems. These systems are capable of detecting specific endocellular targets such as mRNAs, microRNAs or small mols. in real time. In this minireview, we discuss the current progress in the field of DNA-nanoparticles as sensors, their properties, stability, cellular uptake and cytotoxicity.
- 23Seferos, D. S.; Giljohann, D. A.; Hill, H. D.; Prigodich, A. E.; Mirkin, C. A. Nano-flares: Probes for Transfection and mRNA Detection in Living Cells. J. Am. Chem. Soc. 2007, 129, 15477– 15479, DOI: 10.1021/ja077652923https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlKru7jE&md5=71e766b235655781c09ccfc331b37762Nano-Flares: Probes for Transfection and mRNA Detection in Living CellsSeferos, Dwight S.; Giljohann, David A.; Hill, Haley D.; Prigodich, Andrew E.; Mirkin, Chad A.Journal of the American Chemical Society (2007), 129 (50), 15477-15479CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We demonstrate that novel oligonucleotide-modified gold nanoparticle probes hybridized to fluorophore-labeled complements can be used as both transfection agents and cellular "nano-flares" for detecting mRNA in living cells. Nano-flares take advantage of the highly efficient fluorescence quenching properties of gold, cellular uptake of oligonucleotide nanoparticle conjugates without the use of transfection agents, and the enzymic stability of such conjugates, thus overcoming many of the challenges to creating sensitive and effective intracellular probes. Nano-flares exhibit high signaling, have low background fluorescence, and are sensitive to changes in the no. of RNA transcripts present in cells.
- 24Xue, J.; Shan, L.; Chen, H.; Li, Y.; Zhu, H.; Deng, D.; Qian, Z.; Achilefu, S.; Gu, Y. Visual Detection of STAT5B Gene Expression in Living Cell Using the Hairpin DNA Modified Gold Nanoparticle Beacon. Biosens. Bioelectron. 2013, 41, 71– 77, DOI: 10.1016/j.bios.2012.06.06224https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsF2nurnN&md5=3cabf394d58f9bd1d28f2193131d4399Visual detection of STAT5B gene expression in living cell using the hairpin DNA modified gold nanoparticle beaconXue, Jianpeng; Shan, Lingling; Chen, Haiyan; Li, Yang; Zhu, Hongyan; Deng, Dawei; Qian, Zhiyu; Achilefu, Samuel; Gu, YueqingBiosensors & Bioelectronics (2013), 41 (), 71-77CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)Signal transducer and activator of transcription 5B (STAT5B) is an important protein in JAK-STAT signaling pathway that is responsible for the metastasis and proliferation of tumor cells. Detn. of the STAT5B mRNA relating to the STAT5B expression provides insight into the mechanism of tumor progression. In this study, we designed and used a special hairpin DNA for human STAT5B mRNA to functionalize gold nanoparticles, which served as a beacon for detecting human STAT5B expression. Up to 90% quenching efficiency was achieved. Upon hybridizing with the target mRNA, the hairpin DNA modified gold nanoparticle beacons (hDAuNP beacons) release the fluorophores attached at 5' end of the oligonucleotide sequence. The fluorescence properties of the beacon before and after the hybridization with the complementary DNA were confirmed in vitro. The stability of hDAuNP beacons against degrdn. by DNase I and GSH indicated that the prepd. beacon is stable inside cells. The detected fluorescence in MCF-7 cancer cells correlates with the specific STAT5B mRNA expression, which is consistent with the result from PCR measurement. Fluorescence microscopy showed that the hDAuNP beacons internalized in cells without using transfection agents, with intracellular distribution in the cytoplasm rather than the nucleus. The results demonstrated that this beacon could directly provide quant. measurement of the intracellular STAT5B mRNA in living cells. Compared to the previous approaches, this beacon has advantages of higher target to background ratio of detection and an increased resistance to nuclease degrdn. The strategy reported in this study is a promising approach for the intracellular measurement of RNA or protein expression in living cells, and has great potential in the study of drug screening and discovery.
- 25Shi, J.; Zhou, M.; Gong, A. H.; Li, Q. J.; Wu, Q.; Cheng, G. J.; Yang, M. Y.; Sun, Y. C. Fluorescence Lifetime Imaging of Nanoflares for mRNA Detection in Living Cells. Anal. Chem. 2016, 88, 1979– 1983, DOI: 10.1021/acs.analchem.5b0368925https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1KntLw%253D&md5=6344f2d0c3e81690e12cffdf6c60c6a7Fluorescence Lifetime Imaging of Nanoflares for mRNA Detection in Living CellsShi, Jing; Zhou, Ming; Gong, Aihua; Li, Qijun; Wu, Qian; Cheng, Gary J.; Yang, Mingyang; Sun, YaochengAnalytical Chemistry (Washington, DC, United States) (2016), 88 (4), 1979-1983CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)The expression level of tumor-related mRNA can reveal significant information about tumor progression and prognosis, so specific mRNA in cells provides an important approach for biol. and disease studies. Here, fluorescence lifetime imaging of nanoflares in living cells was first employed to detect specific intracellular mRNA. We characterized the lifetime changes of the prepd. nanoflares before and after the treatment of target mRNA and also compared the results with those of fluorescence intensity-based measurements both intracellularly and extracellularly. The nanoflares released the Cy5-modified oligonucleotides and bound to the targets, resulting in a fluorescence lifetime lengthening. This work puts forward another dimension of detecting specific mRNA in cells and can also open new ways for detection of many other biomols.
- 26Halo, T. L.; McMahon, K. M.; Angeloni, N. L.; Xu, Y.; Wang, W.; Chinen, A. B.; Malin, D.; Strekalova, E.; Cryns, V. L.; Cheng, C.; Mirkin, C. A.; Thaxton, C. S. NanoFlares for the Detection,Isolation, and Culture of Live Tumor Cells from Human Blood. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 17104– 17109, DOI: 10.1073/pnas.1418637111There is no corresponding record for this reference.
- 27Prigodich, A. E.; Seferos, D. S.; Massich, M. D.; Giljohann, D. A.; Lane, B. C.; Mirkin, C. A. Nano-flares for mRNA Regulation and Detection. ACS Nano 2009, 3, 2147– 2152, DOI: 10.1021/nn900381427https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXotl2ht7g%253D&md5=85f5a40b73e9de5bb5ce944891f8a987Nano-flares for mRNA regulation and detectionPrigodich, Andrew E.; Seferos, Dwight S.; Massich, Matthew D.; Giljohann, David A.; Lane, Brandon C.; Mirkin, Chad A.ACS Nano (2009), 3 (8), 2147-2152CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)We build off the previously described concept of a nanoflare to develop an oligonucleotide gold nanoparticle conjugate that is capable of both detecting and regulating intracellular levels of mRNA. We characterize the binding rate and specificity of these materials using survivin, a gene assocd. with the diagnosis and treatment of cancer, as a target. The nanoconjugate enters cells and binds mRNA, thereby decreasing the relative abundance of mRNA in a dose- and sequence-dependent manner, resulting in a fluorescent response. This represents the first demonstration of a single material capable of both mRNA regulation and detection. Further, we investigate the intracellular biochem. of the nanoconjugate, elucidating its mechanism of gene regulation. This work is important to the study of biol. active nanomaterials such as the nanoflare and is a first step toward the development of an mRNA responsive "theranostic".
- 28McClellan, S.; Slamecka, J.; Howze, P.; Thompson, L.; Finan, M.; Rocconi, R.; Owen, L. mRNA Detection in Living Cells: A Next Generation Cancer Stem Cell Identification Technique. Methods 2015, 82, 47– 54, DOI: 10.1016/j.ymeth.2015.04.02228https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXntlWntrw%253D&md5=2fc899ba37e9f56c44af074a676b7789mRNA detection in living cells: A next generation cancer stem cell identification techniqueMcClellan, Steven; Slamecka, Jaroslav; Howze, Patrick; Thompson, Lee; Finan, Michael; Rocconi, Rodney; Owen, LaurieMethods (Amsterdam, Netherlands) (2015), 82 (), 47-54CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)Cancer stem cells (CSC) are a distinct subpopulation within a tumor shown to drive tumor progression, metastasis, and recurrence. A review of the literature reveals poor consensus, with the use of a wide variety of surface markers and functional assays to identify and isolate cancer stem cells. Utilizing a novel technol. that enables live-cell mRNA quantitation, we have demonstrated the ability to identify and sort viable CSC based on markers assocd. with stemness in pluripotent cells. Fresh tumor samples from a variety of cancer types were examd. by flow cytometry for Nanog expression. Levels of CSC detected ranged from 6% to 19%. This method of CSC detection was cross-validated with other commonly used surface markers with good correlation. Matrigel invasion assays confirmed that CSC isolated using this method are both highly motile and invasive. This approach simplifies the process of identifying viable CSC from fresh tumor tissue, providing a level of accuracy not previously available. This method may also provide a valuable tool for screening and validating new CSC biomarkers.
- 29Seftor, E. A.; Seftor, R. E. B.; Weldon, D. S.; Kirsammer, G. T.; Margaryan, N. V.; Gilgur, A.; Hendrix, M. J. C. Melanoma Tumor Cell Heterogeneity: A Molecular Approach to Study Subpopulations Expressing the Embryonic Morphogen Nodal. Semin. Oncol. 2014, 41, 259– 266, DOI: 10.1053/j.seminoncol.2014.02.00129https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXns1Cltbs%253D&md5=9b6319f023f4a4d3b573b0d05883b7a5Melanoma Tumor Cell Heterogeneity: A Molecular Approach to Study Subpopulations Expressing the Embryonic Morphogen NodalSeftor, Elisabeth A.; Seftor, Richard E. B.; Weldon, Don S.; Kirsammer, Gina T.; Margaryan, Naira V.; Gilgur, Alina; Hendrix, Mary J. C.Seminars in Oncology (2014), 41 (2), 259-266CODEN: SOLGAV; ISSN:0093-7754. (Elsevier Inc.)A review. As the frequency of melanoma increases, current treatment strategies are struggling to significantly impact patient survival. One of the crit. issues in designing efficient therapies is understanding the compn. of heterogeneous melanoma tumors in order to target cancer stem cells (CSCs) and drug-resistant subpopulations. In this review, we summarize recent findings pertinent to the reemergence of the embryonic Nodal signaling pathway in melanoma and its significance as a prognostic biomarker and therapeutic target. In addn., we offer a novel mol. approach to studying the functional relevance of Nodal-expressing subpopulations and their CSC phenotype.
- 30Li, B. J.; Menzel, U.; Loebel, C.; Schmal, H.; Alini, M.; Stoddart, M. J. Monitoring Live Human Mesenchymal Stromal Cell Differentiation and Subsequent Selection Using Fluorescent RNA-Based Probes. Sci. Rep. 2016, 6, 26014, DOI: 10.1038/srep2601430https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xotlemtb8%253D&md5=cf957f47f61d6bb444b3a32976b98ecaMonitoring live human mesenchymal stromal cell differentiation and subsequent selection using fluorescent RNA-based probesLi, Bojun; Menzel, Ursula; Loebel, Claudia; Schmal, Hagen; Alini, Mauro; Stoddart, Martin J.Scientific Reports (2016), 6 (), 26014CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)Investigating mesenchymal stromal cell differentiation requires time and multiple samples due to destructive endpoint assays. Osteogenesis of human bone marrow derived mesenchymal stromal cells (hBMSCs) has been widely studied for bone tissue engineering. Recent studies show that the osteogenic differentiation of hBMSCs can be assessed by quantifying the ratio of two important transcription factors (Runx2/Sox9). We demonstrate a method to observe mRNA expression of two genes in individual live cells using fluorescent probes specific for Runx2 and Sox9 mRNA. The changes of mRNA expression in cells can be obsd. in a non-destructive manner. In addn., the osteogenic hBMSCs can be prospectively identified and obtained based on the relative intracellular fluorescence of Sox9 in relation to Runx2 using fluorescence activated cell sorting. Relatively homogeneous cell populations with high osteogenic potential can be isolated from the original heterogeneous osteogenically induced hBMSCs within the first week of induction. This offers a more detailed anal. of the effectiveness of new therapeutics both at the individual cell level and the response of the population as a whole. By identifying and isolating differentiating cells at early time points, prospective anal. of differentiation is also possible, which will lead to a greater understanding of MSC differentiation.
- 31Lahm, H.; Doppler, S.; Dressen, M.; Werner, A.; Adamczyk, K.; Schrambke, D.; Brade, T.; Laugwitz, K. L.; Deutsch, M. A.; Schiemann, M.; Lange, R.; Moretti, A.; Krane, M. Live Fluorescent RNA-Based Detection of Pluripotency Gene Expression in Embryonic and Induced Pluripotent Stem Cells of Different Species. Stem Cells 2015, 33, 392– 402, DOI: 10.1002/stem.1872There is no corresponding record for this reference.
- 32Prigodich, A. E.; Randeria, P. S.; Briley, W. E.; Kim, N. J.; Daniel, W. L.; Giljohann, D. A.; Mirkin, C. A. Multiplexed Nanoflares: mRNA Detection in Live Cells. Anal. Chem. 2012, 84, 2062– 2066, DOI: 10.1021/ac202648w32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsVWitbk%253D&md5=8a1084688f99c699ca49314dbc328e66Multiplexed Nanoflares: mRNA Detection in Live CellsPrigodich, Andrew E.; Randeria, Pratik S.; Briley, William E.; Kim, Nathaniel J.; Daniel, Weston L.; Giljohann, David A.; Mirkin, Chad A.Analytical Chemistry (Washington, DC, United States) (2012), 84 (4), 2062-2066CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)We report the development of the multiplexed nanoflare, a nanoparticle agent that is capable of simultaneously detecting two distinct mRNA targets inside a living cell. These probes are spherical nucleic acid (SNA) gold nanoparticle (Au NP) conjugates consisting of densely packed and highly oriented oligonucleotide sequences, many of which are hybridized to a reporter with a distinct fluorophore label and each complementary to its corresponding mRNA target. When multiplexed nanoflares are exposed to their targets, they provide a sequence specific signal in both extra- and intracellular environments. Importantly, one of the targets can be used as an internal control, improving detection by accounting for cell-to-cell variations in nanoparticle uptake and background. Compared to single-component nanoflares, these structures allow one to det. more precisely relative mRNA levels in individual cells, improving cell sorting and quantification.
- 33Pan, W.; Zhang, T.; Yang, H.; Diao, W.; Li, N.; Tang, B. Multiplexed Detection and Imaging of Intracellular mRNAs Using a Four-Color Nanoprobe. Anal. Chem. 2013, 85, 10581– 10588, DOI: 10.1021/ac402700s33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsFGkurjF&md5=a3172b1a65d1a6c5cc252783acc5ff37Multiplexed Detection and Imaging of Intracellular mRNAs Using a Four-Color NanoprobePan, Wei; Zhang, Tingting; Yang, Huijun; Diao, Wei; Li, Na; Tang, BoAnalytical Chemistry (Washington, DC, United States) (2013), 85 (21), 10581-10588CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Simultaneous detection and imaging of multiple intracellular biomarkers hold great promise for early cancer detection. Here, we introduce a four-color nanoprobe that can simultaneously detect and image four types of mRNAs in living cells. The nanoprobe composed of gold nanoparticles functionalized with a dense shell of mol. beacons, which can identify multiple intracellular mRNA transcripts. It shows rapid response, high specificity, nuclease stability, and good biocompatibility. Intracellular expts. indicate that the nanoprobe could effectively distinguish cancer cells from their normal cells, even some mRNAs are overexpressed in normal cells. Moreover, it can identify the changes of the expression levels of mRNA in living cells. The current strategy could provide more-accurate information for early cancer detection and availably avoid false pos. results.
- 34Li, N.; Chang, C.; Pan, W.; Tang, B. A Multicolor Nanoprobe for Detection and Imaging of Tumor-Related mRNAs in Living Cells. Angew. Chem., Int. Ed. 2012, 51, 7426– 7430, DOI: 10.1002/anie.20120376734https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XpsV2lu78%253D&md5=9a17a709aeebd27cd0f92dc8c421cbd5A multicolor nanoprobe for detection and imaging of tumor-related mRNAs in living cellsLi, Na; Chang, Chenyang; Pan, Wei; Tang, BoAngewandte Chemie, International Edition (2012), 51 (30), 7426-7430, S7426/1-S7426/16CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The authors describe a multicolor fluorescence nanoprobe based on nanoflares, which simultaneously detects three intracellular tumor-related mRNAs. The nanoprobe consists of gold nanoparticles (Au NPs) functionalized with a dense shell of recognition sequences (synthetic oligonucleotides) hybridized to three short dye-terminated reporter sequences by gold-thiol bond formation (Figure I). The gold nanoparticles quench the fluorescence of the dyes. The recognition sequences contain 21-base recognition elements for three specific mRNA transcripts: c-myc mRNA, TK1 mRNA, and GalNAc-T mRNA. In the presence of DNA or RNA targets, the recognition sequences hybridize with the complementary target sequences, forming longer and more stable duplexes, causing the release of the reporter sequences, which can then produce fluorescence signals correlated with the relative amt. of the specific DNA or RNA targets.
- 35Heuer-Jungemann, A.; El-Sagheer, A. H.; Lackie, P. M.; Brown, T.; Kanaras, A. G. Selective Killing of Cells Triggered by Their mRNA Signature in the Presence of Smart Nanoparticles. Nanoscale 2016, 8, 16857– 16861, DOI: 10.1039/C6NR06154K35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFWlsLbO&md5=afb6f27c37cec1e3011e74a7c176aa87Selective killing of cells triggered by their mRNA signature in the presence of smart nanoparticlesHeuer-Jungemann, Amelie; El-Sagheer, Afaf H.; Lackie, Peter M.; Brown, Tom; Kanaras, Antonios G.Nanoscale (2016), 8 (38), 16857-16861CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)The design of nanoparticles that can selectively perform multiple roles is of utmost importance for the development of the next generation of nanoparticulate drug delivery systems. So far most research studies are focused on the customization of nanoparticulate carriers to maximize their drug loading, enhance their optical signature for tracking in cells or provide photo-responsive effects for therapeutic purposes. However, a vital requirement of the new generation of drug carriers must be the ability to deliver their payload selectively only to cells of interest rather than the majority of various cells in the vicinity. Here we show for the first time a new design of nanoparticulate drug carriers that can specifically distinguish different cell types based on their mRNA signature. These nanoparticles sense and efficiently kill model tumor cells by the delivery of an anti-cancer drug but retain their payload in cells lacking the specific mRNA target.
- 36Lee, J. M.; Dedhar, S.; Kalluri, R.; Thompson, E. W. The Epithelial-Mesenchymal Transition: New Insights in Signaling, Development, and Disease. J. Cell Biol. 2006, 172, 973– 981, DOI: 10.1083/jcb.20060101836https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjtF2qsLs%253D&md5=5335036255b1f9c9affcf51ed748129dThe epithelial-mesenchymal transition: New insights in signaling, development, and diseaseLee, Jonathan M.; Dedhar, Shoukat; Kalluri, Raghu; Thompson, Erik W.Journal of Cell Biology (2006), 172 (7), 973-981CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)A review. The conversion of an epithelial cell to a mesenchymal cell is crit. to metazoan embryogenesis and a defining structural feature of organ development. Current interest in this process, which is described as an epithelial-mesenchymal transition (EMT), stems from its developmental importance and its involvement in several adult pathologies. Interest and research in EMT are currently at a high level, as seen by the attendance at the recent EMT meeting in Vancouver, Canada (Oct. 1-3, 2005). The meeting, which was hosted by The EMT International Assocn., was the second international EMT meeting, the first being held in Port Douglas, Queensland, Australia in Oct. 2003. The EMT International Assocn. was formed in 2002 to provide an international body for those interested in EMT and the reverse process, mesenchymal-epithelial transition, and, most importantly, to bring together those working on EMT in development, cancer, fibrosis, and pathol. These themes continued during the recent meeting in Vancouver. Discussion at the Vancouver meeting spanned several areas of research, including signaling pathway activation of EMT and the transcription factors and gene targets involved. Also covered in detail was the basic cell biol. of EMT and its role in cancer and fibrosis, as well as the identification of new markers to facilitate the observation of EMT in vivo. This is particularly important because the potential contribution of EMT during neoplasia is the subject of vigorous scientific debate.
- 37Kang, K. A.; Wang, J.; Jasinski, J. B.; Achilefu, S. Fluorescence Manipulation by Gold Nanoparticles: From Complete Quenching to Extensive Enhancement. J. Nanobiotechnol. 2011, 9, 16, DOI: 10.1186/1477-3155-9-1637https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXmtlKkt74%253D&md5=4f85055dd43339debbc0be3c5d24ebceFluorescence manipulation by gold nanoparticles: from complete quenching to extensive enhancementKang, Kyung A.; Wang, Jianting; Jasinski, Jacek B.; Achilefu, SamuelJournal of Nanobiotechnology (2011), 9 (), 16CODEN: JNOAAO; ISSN:1477-3155. (BioMed Central Ltd.)Background: When a fluorophore is placed in the vicinity of a metal nanoparticle possessing a strong plasmon field, its fluorescence emission may change extensively. Our study is to better understand this phenomenon and predict the extent of quenching and/or enhancement of fluorescence, to beneficially utilize it in mol. sensing/imaging. Results: Plasmon field intensities on/around gold nanoparticles (GNPs) with various diams. were theor. computed with respect to the distance from the GNP surface. The field intensity decreased rapidly with the distance from the surface and the rate of decrease was greater for the particle with a smaller diam. Using the plasmon field strength obtained, the level of fluorescence alternation by the field was theor. estd. For exptl. studies, 10 nm GNPs were coated with polymer layer(s) of known thicknesses. Cypate, a near IR fluorophore, was placed on the outermost layer of the polymer coated GNPs, artificially sepd. from the GNP at known distances, and its fluorescence levels were obsd. The fluorescence of Cypate on the particle surface was quenched almost completely and, at approx. 5 nm from the surface, it was enhanced ∼17 times. The level decreased thereafter. Theor. computed fluorescence levels of the Cypate placed at various distances from a 10 nm GNP were compared with the exptl. data. The trend of the resulting fluorescence was similar. The exptl. results, however, showed greater enhancement than the theor. ests., in general. The distance from the GNP surface that showed the max. enhancement in the expt. was greater than the one theor. predicted, probably due to the difference in the two systems. Conclusions: Factors affecting the fluorescence of a fluorophore placed near a GNP are the GNP size, coating material on GNP, wavelengths of the incident light and emitted light and intrinsic quantum yield of the fluorophore. Exptl., we were able to quench and enhance the fluorescence of Cypate, by changing the distance between the fluorophore and GNP. This ability of artificially controlling fluorescence can be beneficially used in developing contrast agents for highly sensitive and specific optical sensing and imaging.
- 38Wu, Z. S.; Jiang, J. H.; Fu, L.; Shen, G. L.; Yu, R. Q. Optical Detection of DNA Hybridization Based on Fluorescence Quenching of Tagged Oligonucleotide Probes by Gold Nanoparticles. Anal. Biochem. 2006, 353, 22– 29, DOI: 10.1016/j.ab.2006.01.018There is no corresponding record for this reference.
- 39Zheng, D.; Seferos, D. S.; Giljohann, D. A.; Patel, P. C.; Mirkin, C. A. Aptamer Nano-flares for Molecular Detection in Living Cells. Nano Lett. 2009, 9, 3258– 3261, DOI: 10.1021/nl901517b39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXptlSmtbw%253D&md5=ed0a131b9ccca7002b24570f3032628bAptamer Nano-flares for Molecular Detection in Living CellsZheng, Dan; Seferos, Dwight S.; Giljohann, David A.; Patel, Pinal C.; Mirkin, Chad A.Nano Letters (2009), 9 (9), 3258-3261CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The authors demonstrate a composite nanomaterial, termed an aptamer nano-flare, that can directly quantify an intracellular analyte in a living cell. Aptamer nano-flares consist of a gold nanoparticle core functionalized with a dense monolayer of nucleic acid aptamers with a high affinity for ATP. The probes bind selectively to target mols. and release fluorescent reporters which indicate the presence of the analyte. Addnl., these nanoconjugates are readily taken up by cells where their signal intensity can be used to quantify intracellular analyte concn. These nanoconjugates are a promising approach for the intracellular quantification of other small mols. or proteins, or as agents that use aptamer binding to elicit a biol. response in living systems.
- 40Gilleron, J.; Querbes, W.; Zeigerer, A.; Borodovsky, A.; Marsico, G.; Schubert, U.; Manygoats, K.; Seifert, S.; Andree, C.; Stoeter, M.; Epstein-Barash, H.; Zhang, L.; Koteliansky, V.; Fitzgerald, K.; Fava, E.; Bickle, M.; Kalaidzidis, Y.; Akinc, A.; Maier, M.; Zerial, M. Image-Based Analysis of Lipid Nanoparticle-Mediated siRNA Delivery, Intracellular Trafficking and Endosomal Escape. Nat. Biotechnol. 2013, 31, 638– 646, DOI: 10.1038/nbt.261240https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXpvVymtbg%253D&md5=6fe33f7714c778659e9f87a38808ea9eImage-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escapeGilleron, Jerome; Querbes, William; Zeigerer, Anja; Borodovsky, Anna; Marsico, Giovanni; Schubert, Undine; Manygoats, Kevin; Seifert, Sarah; Andree, Cordula; Stoeter, Martin; Epstein-Barash, Hila; Zhang, Ligang; Koteliansky, Victor; Fitzgerald, Kevin; Fava, Eugenio; Bickle, Marc; Kalaidzidis, Yannis; Akinc, Akin; Maier, Martin; Zerial, MarinoNature Biotechnology (2013), 31 (7), 638-646CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Delivery of short interfering RNAs (siRNAs) remains a key challenge in the development of RNA interference (RNAi) therapeutics. A better understanding of the mechanisms of siRNA cellular uptake, intracellular transport and endosomal release could critically contribute to the improvement of delivery methods. Here we monitored the uptake of lipid nanoparticles (LNPs) loaded with traceable siRNAs in different cell types in vitro and in mouse liver by quant. fluorescence imaging and electron microscopy. We found that LNPs enter cells by both constitutive and inducible pathways in a cell type-specific manner using clathrin-mediated endocytosis as well as macropinocytosis. By directly detecting colloidal-gold particles conjugated to siRNAs, we estd. that escape of siRNAs from endosomes into the cytosol occurs at low efficiency (1-2%) and only during a limited window of time when the LNPs reside in a specific compartment sharing early and late endosomal characteristics. Our results provide insights into LNP-mediated siRNA delivery that can guide development of the next generation of delivery systems for RNAi therapeutics.
- 41Blanco, E.; Shen, H.; Ferrari, M. Principles of Nanoparticle Design for Overcoming Biological Barriers to Drug Delivery. Nat. Biotechnol. 2015, 33, 941– 951, DOI: 10.1038/nbt.333041https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVymt73I&md5=d693b4429d7ad4cc8b3898f7a0fb6235Principles of nanoparticle design for overcoming biological barriers to drug deliveryBlanco, Elvin; Shen, Haifa; Ferrari, MauroNature Biotechnology (2015), 33 (9), 941-951CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Biol. barriers to drug transport prevent successful accumulation of nanotherapeutics specifically at diseased sites, limiting efficacious responses in disease processes ranging from cancer to inflammation. Although substantial research efforts have aimed to incorporate multiple functionalities and moieties within the overall nanoparticle design, many of these strategies fail to adequately address these barriers. Obstacles, such as nonspecific distribution and inadequate accumulation of therapeutics, remain formidable challenges to drug developers. A reimagining of conventional nanoparticles is needed to successfully negotiate these impediments to drug delivery. Site-specific delivery of therapeutics will remain a distant reality unless nanocarrier design takes into account the majority, if not all, of the biol. barriers that a particle encounters upon i.v. administration. By successively addressing each of these barriers, innovative design features can be rationally incorporated that will create a new generation of nanotherapeutics, realizing a paradigmatic shift in nanoparticle-based drug delivery.
- 42Perez-Arnaiz, C.; Busto, N.; Leal, J. M.; Garcia, B. New Insights into the Mechanism of the DNA/Doxorubicin Interaction. J. Phys. Chem. B 2014, 118, 1288– 1295, DOI: 10.1021/jp411429g42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXnsV2qtQ%253D%253D&md5=65a09adebe62d60071b731847caed1a1New Insights into the Mechanism of the DNA/Doxorubicin InteractionPerez-Arnaiz, Cristina; Busto, Natalia; Leal, Jose M.; Garcia, BegonaJournal of Physical Chemistry B (2014), 118 (5), 1288-1295CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)Doxorubicin (DOX) is an important anthracycline antibiotic whose intricate features of binding to DNAs, not yet fully understood, have been the object of intense debate. The dimerization equil. has been studied at pH = 7.0, I = 2.5 mM, and T = 25°. A thermodn. and kinetic study of the binding of doxorubicin to DNA, carried out by CD, viscometry, differential scanning calorimetry, fluorescence, isothermal titrn. calorimetry, and T-jump relaxation measurements, has enabled us to characterize for the first time two different types of calf thymus DNA (ctDNA)/DOX complexes: PD1 for CDOX/CDNA < 0.3, and PD2 for higher drug content. The nature of the PD1 complex is described better in light of the affinity of DOX with the synthetic copolymers [poly(dA-dT)]2 and [poly(dG-dC)]2. The formation of PD1 has been categorized kinetically as a two-step mechanism in which the fast step is the groove binding in the AT region, and the slow step is the intercalation into the GC region. This bifunctional nature provides a plausible explanation for the high PD1 const. obtained (K1 = 2.3 × 108 M-1). Moreover, the formation of an external aggregate complex ctDNA/DOX (PD2) at the expense of PD1, with K2 = 9.3 × 105 M-1, has been evinced.
- 43Hajihassan, Z.; Rabbani-Chadegani, A. Studies on the Binding Affinity of Anticancer Drug Mitoxantrone to Chromatin, DNA and Histone Proteins. J. Biomed. Sci. 2009, 16, 31, DOI: 10.1186/1423-0127-16-3143https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1M3jtVanuw%253D%253D&md5=9dd75cc4aaccdbf234b8d5071cc4606cStudies on the binding affinity of anticancer drug mitoxantrone to chromatin, DNA and histone proteinsHajihassan Zahra; Rabbani-Chadegani AzraJournal of biomedical science (2009), 16 (), 31 ISSN:.Mitoxantrone is a potent antitumor drug, widely used in the treatment of various cancers. In the present study, we have investigated and compared the affinity of anticancer drug, mitoxantrone, to EDTA-soluble chromatin (SE-chromatin), DNA and histones employing UV/Vis, fluorescence, CD spectroscopy, gel electrophoresis and equilibrium dialysis techniques. The results showed that the interaction of mitoxantrone with SE-chromatin proceeds into compaction/aggregation as revealed by reduction in the absorbencies at 608 and 260 nm (hypochromicity) and disappearance of both histones and DNA on the gels. Mitoxantrone interacts strongly with histone proteins in solution making structural changes in the molecule as shown by CD and fluorescence analysis. The binding isotherms demonstrate a positive cooperative binding pattern for the chromatin- mitoxantrone interaction. It is suggested higher binding affinity of mitoxantrone to chromatin compared to DNA implying that the histone proteins may play an important role in the chromatin- mitoxantrone interaction process.
- 44Ohnuma, T.; Matsumoto, T.; Itoi, A.; Kawana, A.; Nishiyama, T.; Ogura, K.; Hiratsuka, A. Enhanced Sensitivity of A549 Cells to the Cytotoxic Action of Anticancer Drugs via Suppression of Nrf2 by Procyanidins from Cinnamomi Cortex Extract. Biochem. Biophys. Res. Commun. 2011, 413, 623– 629, DOI: 10.1016/j.bbrc.2011.09.014There is no corresponding record for this reference.
Supporting Information
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b08620.
Additional experimental detail and data including nanoparticle characterization (TEM, DLS, melting curves, nuclease assays), detailed oligonucleotide sequences and further intracellular studies (confocal images, TEM cross sectioning, immunofluorescent labeling, RT-qPCR); the raw data for this work can be found at http://doi.org/10.5258/SOTON/D0448 (PDF)
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