A Markedly Improved Synthetic Approach for the Preparation of

Subscriber access provided by University of Sunderland

Article

A Markedly Improved Synthetic Approach for the Preparation of Multifunctional Au-DNA Nanoparticle Conjugates Modified with Optical and MR Imaging Probes Matthew W Rotz, Robert J Holbrook, Keith W MacRenaris, and Thomas J. Meade Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00504 • Publication Date (Web): 07 Sep 2018 Downloaded from http://pubs.acs.org on September 9, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

A Markedly Improved Synthetic Approach for the Preparation of Multifunctional Au-DNA Nanoparticle Conjugates Modified with Optical and MR Imaging Probes Matthew W. Rotz, Robert J. Holbrook, Keith W. MacRenaris, Thomas J. Meade* Department of Chemistry, Molecular Biosciences, Neurobiology, Biomedical Engineering, and Radiology, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113, United States. Corresponding Author: [email protected] Abstract: We describe a new, and vastly superior approach for labeling spherical nucleic acid conjugates (SNAs) with diagnostic probes. SNAs have been shown to provide the unique ability to traverse the cell membrane and deliver surface conjugated DNA into cells while preserving the DNA from nuclease degradation. Our previous work on preparing diagnostically labeled SNAs was labor intensive, relatively low yielding and costly. Here, we describe a straightforward and facile preparation for labeling SNAs with optical and MR imaging probes with significantly improved physical properties. The synthesis of Gd(III) labeled DNA Au nanoparticle conjugates is achieved by sequential conjugation of 3’-thiol-modified oligonucleotides and co-functionalization of the particle surface with the subsequent addition of 1,2 diothiolate modified chelates of Gd(III) (abbreviated: DNA-GdIII@AuNP). This new generation of SNA conjugates has a 2-fold increase of DNA labeling and a 1.4-fold increase in Gd(III) loading compared to published constructs. Furthermore, the relaxivity (r1) is observed to increase 4.5-fold compared to the molecular dithiolane-Gd(III) complex, and 1.4-fold increase relative to previous particle constructs where the Gd(III) complexes were conjugated to the oligonucleotides rather than directly to the Au particle. Importantly, this simplified approach (2 steps) exploits the advantages of previous Gd(III) labeled SNA platforms, however this new approach is scalable,

ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

eliminates modification of DNA for attaching the contrast agent, and the particles exhibit improved cell labeling. ___ For nearly two decades spherical nucleic acid conjugates (SNAs) have been prepared for applications ranging from the engineering of supra-molecular lattices, monitoring gene expression and regulation, bio-detection, as cell transfection agents, and more recently for cancer therapy.1-11 These nanoparticle conjugates have been thoroughly investigated with respect to their in vivo stability and toxicity profiles, hybridization thermodynamics, kinetics, and nuclease resistance in vitro.12-17 As the clinical community has become more focused on understanding diseases at the molecular and cellular level, a fundamental need for noninvasive methods of studying in vivo biochemical processes has emerged.18-21 Traditional methods such as Western blotting and immunohistochemistry require sample destruction that prevents longitudinal studies. To overcome these limitations a variety of molecular imaging techniques have been developed to investigate in vivo events, making use of such modalities as optical, MRI, PET, SPECT and ultrasound.20, 22-27 Each of these modalities has unique advantages and disadvantages regarding sensitivity, spatial/temporal resolution, and the type of radiation or lack thereof. The in vivo detection limits of each modality can be augmented by using contrast agents and reporter probes and are used in applications such as cell tracking, reporting gene expression, and monitoring therapeutic paradigms.28-40 MR imaging is a staple of experimental and clinical diagnostic radiology due to excellent soft-tissue contrast, high imaging resolution, and the absence of ionizing radiation. MRI is capable of 3D-imaging of biological structures, and in research settings at very high field,


Page 2 of 18

Page 3 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


processes can be imaged at near-cellular resolution (~10 um).20 Unlike fluorescence and other light-based imaging techniques, MRI does not require optically transparent samples. Detailed structural information can be obtained in minutes, and single slices in seconds. Due to its established position as a clinical diagnostic tool, MR imaging is a major focus of translational imaging research. Two fundamental challenges in the development MR contrast agents (CA’s) that must be overcome for use in experimental preclinical imaging are: i. amplification of the contrast agent signal; ii. specific agent delivery to cells and tissues of interest. The majority of CA’s are based on either paramagnetic or superparamagnetic metal ions. These species act as potent relaxation agents due to the presence of unpaired electrons. In general, paramagnetic (T1) agents, such as Gd(III), decrease T1 with a lesser effect on T2, resulting in increased signal in the vicinity of the agent. Conversely, superparamagnetic agents primarily decrease the T2 with a lesser effect on T1, resulting in a signal void in the vicinity of the agent. Gd(III) is the most widely used metal ion for T1 CA’s due to its high magnetic moment and long electron relaxation time. To overcome these challenges we have developed a number of nanoparticle conjugates that significantly enhance the observed MR signal (1000 fold) over molecular complexes.40-45 This synthetic strategy consisted of covalently attaching Gd(III) complexes to thiol-modified oligonucleotides (via click chemistry) and conjugating this derivative to 13.1 nm gold nanoparticles (AuNPs).5, 42 Because of the increased local concentration of Gd(III) per particle, there is a significant increase in the observed r1 (relaxivity mM-1s-1, particularly at low magnetic field strengths).5 As a result of this MR signal amplification we have successfully detected MR reporter genes40, imaged the vivo biodistribution of glioblastoma17 and fate mapped human neural stem cells.44



Page 4 of 18

These promising results stimulated the design of the next generation of Gd(III)-modified SNAs while maintaining the modularity and utility of these important nanoparticles. Our goal in this study was to significantly improve two important features: i.) further increase the Gd(III) payload through direct surface conjugation to the AuNP and ii.) greatly simplify the synthesis by eliminating the Gd(III) attachment to the SH- modified oligonucleotides.

RESULTS AND DISCUSSION Here, we describe a straightforward and scalable approach to the synthesis of a 1,2 dithiolane modified Gd(III)-macrocycle (4: See Supporting Information for details), conjugated to a SNA to form dt-Gd(III)-SNA (Figure 1). We characterized these new constructs by measuring the proton relaxation properties, particle stability, and quantify cell-labeling efficiency.

dt-Gd(III)-SNA

3’ HS – TTT – TTT – TTT – TTT – TTT – Cy3-5’

5

4

Figure 1. Synthetic Strategy for the preparation of dt-Gd(III)-SNA nanoparticles: Synthetic scheme of dt-Gd(III) particle conjugates is achieved without the presence of reducing agents. The synthesis and characterization of 4 and 5 can be found in Supporting Information. Standard salt aging conjugation protocol using 5’Cy3 poly-dT SNAs starting from citrate-stabilized AuNPs. Once the 5’Cy3 poly-dT SNAs are conjugated to the AuNP, complex 4 is added to form the final product dt-Gd(III)-SNA.


Page 5 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In a systematic study by Mirkin et al. to determine the maximum achievable density of thiolated oligonucleotides that could be loaded onto a AuNP surface, 15 nm spherical particles were functionalized with densities up to 19 pmol/cm2 when salt-aged with 1M sodium chloride (corresponding to 0.11 DNA/nm2, or 78 DNA/AuNP).2,

3

It is known that the density of 3’-

thiolated DNA packing on a spherical AuNP surface is limited by the negative charge present on the polyanionic backbone of immobilized oligonucleotides.46 By comparison, organic ligand densities have been reported to reach as high as 6.3 ± 0.6 monothiolated ligands per nm2 for AuNPs of comparable diameter (3-mecaptopropionic acid).47 Given the greater than 50-fold difference between oligonucleotide and organic ligand densities achievable on the particle surface, we estimated that the surface area remaining unmodified (after saturation of the particle with thiolated oligonucleotides) could support a densely packed layer of Gd(III) complexes (an approach that we refer to as backfilling because the Gd(III) is added post DNA addition). Importantly, dt-Gd(III)-SNAs have demonstrated the ability to bind previously synthesized SNAs at high surface densities, and can be modified (i.e., backfilled with 4) onto the surface of these particles without the need for reducing agents. In addition, this nanoconjugate design is observed to increase r1 relaxivity per Gd(III), and particle payload by greater than 3fold when compared to the previously reported DNA-GdIII@AuNPs, while maintaining high stability, biocompatibility and cellular uptake of previous generations. This new dt-Gd(III)-SNA design leverages numerous beneficial features of previous Gd(III) nanoconjugate CAs5, 42, while providing an extremely facile synthesis (and scalability) of other Gd(III)-labeled particle conjugates.48-50 The 1,2-dithiolane anchor was chosen for this work because it has been found that the cyclic disulfide functionality is an excellent surface binding ligand for gold.51-54 Significantly, studies have described an enhancement of colloidal stability



using DNA modified with lipoic acid as compared to mono-thiolated DNAs in the presence of high concentrations of dithiothreitol.55 The dt-Gd(III) complex used here (4, Figure 1) was synthesized by using the Cu(I)-catalyzed Huisgen azide-alkyne cycloaddition of an azide derivative of (±)-lipoic acid, and a previously published alkyne bearing, τm-optimized Gd(III) (complex 3, See SI).56 Purification of dt-Gd(III) was performed using HPLC and characterization confirmed by high resolution ESI-TOF mass spectroscopy (Schemes S1-3, Figure S1). To determine the maximum density of Gd(III) complexes on the surface of the particle, 13 nm AuNPs were functionalized with only 4 for quantification of surface loading and r1 relaxivity. Particle conjugation was achieved by mixing an excess of 4 into a stock of freshly prepared solution of 13.0 ± 1.6 nm citrate stabilized AuNPs with 0.01% tween with shaking for 24 hours. After labeling, the particles were washed and concentrated by centrifugation. The pure 4 particle conjugates have a r1 of 17.1 mM-1 s-1 at 37 °C (1.41 T), and a total surface loading of 1125 ± 17 Gd(III) complexes per particle (determined by inductively coupled plasma mass spectrometry ICP-MS). To test the efficacy of this approach, poly-dT-SNAs were synthesized using the same batch of 13 nm citrate-stabilized AuNPs as used to prepare the pure 4 conjugates. Specifically, SNAs were made by salt-aging 3’-thiolated 24-mer poly-dT DNA bearing a 5’ Cy3 fluorophore. Upon completion of the conjugation process, particles were concentrated and purified from excess DNA, citrate, and sodium chloride by successive rounds of centrifugation. To assess the DNA conjugation efficiency, SNAs were incubated with potassium cyanide to digest the gold cores, and UV/Vis measurement of Cy3 was used to quantify DNA loading. Results indicated a total surface loading of 199 ± 0.6 strands per particle.57 Using the purified poly-dT SNAs


Page 6 of 18

Page 7 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


concentrated to ten times their initial concentration, 50,000 equivalents of dt-Gd(III) were added directly, and functionalization was achieved with stirring over 24 hours. This “backfilling” approach to prepare dt-Gd(III)-SNAs proceeded without the use of any reducing agents,58, 59 and were purified by centrifugation with an r1 of 22.0 mM-1 s-1 at 37 °C (1.41 T), with a particle payload of 730 ± 11 Gd(III) complexes per particle (Table 1). Relative to the pure dt-Gd(III) conjugates, this represents a 23% improvement in r1, sacrificing only 35% of the Gd(III) loading. Having removed a portion of the initial poly-dT conjugation mixture, dtGd(III) was added directly (without purification of particles from excess DNA) to test the capacity of performing DNA conjugation and backfilling is a single vessel. After a further 24 hours to complete the addition of 4, particles were purified by centrifugation. The cofunctionalization process produced dt-Gd(III)-SNAs loaded with 691 dt-Gd(III) per particle, which is in good agreement with previous preparations of dt-Gd(III)-SNAs and confirms the effectiveness of combining these conjugation steps. Furthermore, the kinetics of the backfilling procedure was examined. A purified batch of poly-dT SNAs were mixed with dt-Gd(III), and aliquots were removed at 1, 2, 4, 8 and 24 hours. Particles from each time point were purified and results indicated that 76% of particle loading was complete within 60 minutes and completed after 24 hours (See Figure S10). To compare how the new dt-Gd(III)-SNAs perform relative to the previous generations of Gd(III)-DNAs AuNP conjugates (in which the Gd(III) is covalently attached to the DNA), GdDNA@spheres were synthesized as recently described.41 Analysis of these conjugates showed increased particle loading compared with all previously reported 13 nm nanoconjugates of this design: totaling 515 ± 15 Gd(III) complexes per particle and an r1 of 12.8 mM-1 s-1 at 37 °C (1.41 T). Despite the remarkably high Gd(III) loading observed for the Gd-DNA@spheres, the density



of Gd(III) was still limited to only 0.97 complexes per nm2 resulting from 103 ± 3 DNA per particle (corresponding to a DNA density of 0.19 DNA per nm2). The limitations which prevented further loading result from density restrictions supplied by the negative charges on the DNA backbone, and the steric crowding imposed by the covalent linker from the modified DNA bases to Gd(III) complexes.5 In comparison, the loading densities of Gd(III) and DNA for dtGd(III)-SNAs were 1.4- and 2-fold greater, respectively (Table S16). Importantly, the observed increase in density of oligonucleotide loading may provide both increased utility for sequences tailored to specific applications, and enhanced nuclease stability (relative to less densely functionalized SNAs).3 Table 1 Relaxivity r1 and particle loading for the Gd(III) nanoconjugates reported. Ionic relaxivity describes the relaxivity per Gd(III) ion. Molecular relaxivity refers to the sum of relaxivities/particle.

r1 relaxivity (mM-1 s-1)a Particle ionic molecular Loading dt-Gd(III) 4.9 NA NA dt-Gd(III) pure particle 17.1 19270 1125 ± 17 Gd-DNA@spheresb 12.8 6620 515 ± 15 dt-Gd(III)-SNA’s 22.0 16080 730 ± 11 a 60 MHz, 37 °C in water with 0.01% tween 20: bPrepared as as previously described42: NA = not applicable. To investigate the stability of the 1,2-dithiolane conjugates on the Au particle, the loss of 4 from both dt-Gd(III)-SNA and Gd-DNA@sphere constructs in cell culture conditions were compared using ICP-MS. Specifically, concentrated solutions of both conjugates were diluted to equimolar concentrations in PBS, DMEM, 10% FBS in DMEM and 100% FBS, and incubated at 37 °C under sterile conditions for two weeks. Particle concentration and Gd(III) loading was quantified at time zero for each solution, and each condition was run in duplicate. Time points were recorded at each of 24, 48, 120, 168, and 336 hours by removal of an aliquot from the incubated solution followed by centrifugation and ICP-MS analysis of supernatant Gd(III).


Page 8 of 18

Page 9 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In addition, the colloidal stability was assessed by observation of the plasmon resonance peak at time points of 1, 7 and 14 days for both constructs under all conditions. After 24 hours, both nanoconjugates had visibly precipitated out of DMEM, while the remaining solutions remained visibly red and colloidally stable throughout all time points (Figure 2a). Analysis of the surface plasmon resonance peak for each construct indicated the maximum value at 527 and 521 nm for backfilled and Gd(III) DNA conjugated particles in water for time zero, and all solutions showed a variable increase in this value over 14 days, particularly solutions of FBS (Figure S11). Results of the supernatant analysis were plotted as a percentage of loss from the starting Gd(III) concentration (Figure 2b). These data indicate that under these stringent conditions, both constructs were vulnerable to degradation under all conditions excepting PBS, despite the different presentation and number of thiols present on the monothiolated Gd(III) DNAs and the backfilled dt-Gd(III) complexes. However, under commonly used cell culture conditions of 10 % FBS with an incubation time of 24 hours, the new dt-Gd(III)-SNAs were 2-times more stable than the Gd-DNA@spheres with a Gd(III) loss of 12.9 %. Given the similar trend of the degradation observed between the two constructs, it remains unclear the nature of coordination of 4 to the gold surface of the particle and its role in stability, relative to monothiolated DNA. To answer this question, experiments are underway to verify the degree in which the 1,2-dithiolane is bound in a bidentate fashion to the Au surface.



a) Poly T Backfilled AuNP in PBS

Poly T Backfilled AuNP in 10% FBS

0.8 0.6 0.4 0.2 0.0 450

500

550

600

650

1.0 0.8 0.6 0.4 0.2 0.0 450

500

wavelength (nm)

b)

550

600

650

time 0 1 day 7 days 14 days water control

0.8 0.6 0.4 0.2 0.0 450

500

Backfilled Poly T AuNP

550

600

650

wavelength (nm)

FBS Supernatant Analysis

Gd-DNA AuNP


100

Gd-DNA AuNP

80

Gd loss (%)

80

Gd loss (%)

1.0

wavelength (nm)

10% FBS Supernatant Analysis 100

Poly T Backfilled AuNP in FBS Normalize d Absorbance (AU)

1.0

Normalize d Absorbance (AU)

Normalize d Absorbance (AU)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 18

60 40 20

60 40 20

0

0 0

100

200

300

400

0

100

Time (h)

200

300

400

Time (h)

Figure 2. Stability of dt-Gd(III)-SNAs measured under various cell culture related conditions over 14 days at 37 °C. a) dt-Gd(III)-SNAs were examined for colloidal stability in PBS, 10% FBS and FBS. b) Supernatant analysis of Gd(III) loss under similar conditions in 10% FBS and 100% FBS. Gd(III) loss during long-term storage in PBS was minimal (Figure S12). To test the biocompatibility and cellular uptake efficiency of dt-Gd(III)-SNAs and GdDNA@spheres, HeLa cells were prepared and incubated using various dilutions of nanoconjugates in 10% FBS in DMEM. After 24 hours cells were washed, trypsinized, and counted. Cell counts were as expected and no cell toxicity was observed for any of the incubation concentrations. Counted cells were digested in 1:1 HNO3 and HCl and Gd(III) and gold content was examined using ICP-MS. Per concentration of nanoconjugates observed, the dtGd(III)-SNAs provided improved Gd(III) uptake, particularly at lower incubation concentrations (Figure 3). Interestingly, the improved Gd(III) payload was not the only cause of the improved uptake, as cells dosed with the dt-Gd(III)-SNAs appeared to take up more of the nanoconjugates at each of the concentrations examined (Figure S13). This result can be explained by the higher


Page 11 of 18

density loading of DNA present on the dt-Gd(III)-SNAs relative to the DNA-Gd@spheres, which are limited in the densities of DNA which can be achieved due to the steric crowding of the particle surface resultant from the long linker arm of the covalently attached Gd(III) complex.3

a

b 24 h Cellular Uptake of Gd(III)-SNA Conjugates Backfilled poly dT SNAs

24 h Cellular Uptake of Gd(III)-SNA Conjugates

Gd(III)-DNA SNAs

Backfilled poly dT SNAs

Gd(III)-DNA SNAs

0.5

fmol Gd(III) per cell

0.5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.4 0.3 0.2 0.1 0.0

0.4 0.3 0.2 0.1 0.0

0

20

40

60

Gd(III) Dosing Concentration ( µM)

0

50

100

150

AuNP Dosing Concentration (nM)

Figure 3. 24 hour cell uptake experiment comparing dt-Gd(III)-SNAs and DNA-Gd@spheres described by (a) Gd(III) dosing, or (b) AuNP dosing concentrations.

CONCLUSION We have discovered a facile and scalable methodology for the functionalization of SNA’s with optical and Gd(III) MR imaging probes. The nanoconjugates consist of an optical probe (Cy3labled-DNA: 5) and hundreds of MR contrast agents (dithiolane functionalized Gd(III) complexes: 4) to create a powerful multimodal imaging conjugate. The new class of particles exhibits increased r1 relaxivity, improved Gd(III) loading/particle, increased cellular uptake at lower incubations concentrations, with comparable conjugate stability relative to the previous generations of our multimodal nanoparticles.41-43 The observed increase in low-field magnetic relaxivity, improved Gd(III) loading, and significantly higher cellular uptake of the particles will allow the use of far less contrast agent per cell labeling experiments. Over the last two decades



we have routinely discovered that there is no “one imaging probe fits” for different cell types. Due to the modular synthesis of the SNAs, many different nucleotide sequences can be tested and targeting groups can replace the Cy3 dye on the 5’ end of the DNA. This vastly simplified synthetic approach is facilitating the creation of new Gd(III) labeled nanoconjugates in our laboratory that are tailored to each cell type. By the use of this the new “backfilling” approach for the preparation of multimodal imaging probes we can quickly learn which probe(s) are best suited to each individual cell type. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

17.

18.

N. L. Rosi, D. A. Giljohann, C. S. Thaxton, A. K. R. Lytton-Jean, M. S. Han and C. A. Mirkin, Science, 2006, 312, 1027-1030. S. J. Hurst, A. K. Lytton-Jean and C. A. Mirkin, Anal Chem, 2006, 78, 8313-8318. D. A. Giljohann, D. S. Seferos, P. C. Patel, J. E. Millstone, N. L. Rosi and C. A. Mirkin, Nano Letters, 2007, 7, 3818-3821. P. C. Patel, D. A. Giljohann, W. L. Daniel, D. Zheng, A. E. Prigodich and C. A. Mirkin, Bioconjug Chem, 2010, 21, 2250-2256. Y. Song, X. Xu, K. W. MacRenaris, X.-Q. Zhang, C. A. Mirkin and T. J. Meade, Angewandte Chemie International Edition, 2009, 48, 9143-9147. B. Bo, T. Zhang, Y. Jiang, H. Cui and P. Miao, Analytical Chemistry, 2018, 90, 2395-2400. G. Han, N. S. Chari, A. Verma, R. Hong, C. T. Martin and V. M. Rotello, Bioconjugate Chemistry, 2005, 16, 1356-1359. B. L. Fay, J. R. Melamed and E. S. Day, International Journal of Nanomedicine, 2015, 10, 69316941. N. S. Abadeer and C. J. Murphy, The Journal of Physical Chemistry C, 2016, 120, 4691-4716. J. Mosquera, M. Henriksen-Lacey, I. García, M. Martínez-Calvo, J. Rodríguez, J. L. Mascareñas and L. M. Liz-Marzán, Journal of the American Chemical Society, 2018, 140, 4469-4472. M. Sanromán-Iglesias, C. H. Lawrie, L. M. Liz-Marzán and M. Grzelczak, ACS Applied Nano Materials, 2018, 1, 3741-3746. J. I. Cutler, E. Auyeung and C. A. Mirkin, J Am Chem Soc, 2012, 134, 1376-1391. F. Li, H. Zhang, B. Dever, X. F. Li and X. C. Le, Bioconjug Chem, 2013, 24, 1790-1797. D. S. Seferos, A. E. Prigodich, D. A. Giljohann, P. C. Patel and C. A. Mirkin, Nano Lett, 2009, 9, 308-311. W. E. Briley, M. H. Bondy, P. S. Randeria, T. J. Dupper and C. A. Mirkin, Proc Natl Acad Sci U S A, 2015, 112, 9591-9595. T. L. Halo, K. M. McMahon, N. L. Angeloni, Y. Xu, W. Wang, A. B. Chinen, D. Malin, E. Strekalova, V. L. Cryns, C. Cheng, C. A. Mirkin and C. S. Thaxton, Proc Natl Acad Sci U S A, 2014, 111, 1710417109. S. A. Jensen, E. S. Day, C. H. Ko, L. A. Hurley, J. P. Luciano, F. M. Kouri, T. J. Merkel, A. J. Luthi, P. C. Patel, J. I. Cutler, W. L. Daniel, A. W. Scott, M. W. Rotz, T. J. Meade, D. A. Giljohann, C. A. Mirkin and A. H. Stegh, Sci Transl Med, 2013, 5, 209ra152. J. L. Major and T. J. Meade, Acc Chem Res, 2009, 42, 893-903.


Page 12 of 18

Page 13 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.

46. 47. 48.

L. M. Manus, D. J. Mastarone, E. A. Waters, X. Q. Zhang, E. A. Schultz-Sikma, K. W. Macrenaris, D. Ho and T. J. Meade, Nano Lett, 2010, 10, 484-489. M. L. James and S. S. Gambhir, Physiol Rev, 2012, 92, 897-965. A. T. Preslar, G. Parigi, M. T. McClendon, S. S. Sefick, T. J. Moyer, C. R. Haney, E. A. Waters, K. W. MacRenaris, C. Luchinat, S. I. Stupp and T. J. Meade, ACS Nano, 2014, 8, 7325-7332. K. W. Ferrara, Adv Drug Deliv Rev, 2008, 60, 1097-1102. L. Abou-Elkacem, S. V. Bachawal and J. K. Willmann, Eur J Radiol, 2015, 84, 1685-1693. X. Sun, W. Cai and X. Chen, Acc Chem Res, 2015, 48, 286-294. S. M. Ametamey, M. Honer and P. A. Schubiger, Chem Rev, 2008, 108, 1501-1516. E. Terreno, D. D. Castelli, A. Viale and S. Aime, Chem Rev, 2010, 110, 3019-3042. A. Taruttis, G. M. van Dam and V. Ntziachristos, Cancer Res, 2015, 75, 1548-1559. A. Datta and K. N. Raymond, Acc Chem Res, 2009, 42, 938-947. A. Merbach and E. Toth, The Chemistry of Contrast Agents in Medical Magnetic Resonance Imaging, John Wiley and Sons, LTD, Chichester, 2001. J. L. Major, G. Parigi, C. Luchinat and T. J. Meade, Proc Natl Acad Sci U S A, 2007, 104, 1388113886. E. Boros, E. M. Gale and P. Caravan, Dalton transactions, 2015, 44, 4804-4818. J. Garcia, T. Tang and A. Y. Louie, Nanomedicine (Lond), 2015, 10, 1343-1359. U. Seibold, B. Wangler, R. Schirrmacher and C. Wangler, Biomed Res Int, 2014, 2014, 153741. J. A. Thomas, Chem Soc Rev, 2015, 44, 4494-4500. K. W. MacRenaris, Z. Ma, R. L. Krueger, C. E. Carney and T. J. Meade, Bioconjug Chem, 2016, 27, 465-473. V. S. Harrison, C. E. Carney, K. W. MacRenaris, E. A. Waters and T. J. Meade, J Am Chem Soc, 2015, 137, 9108-9116. I. Minn, A. Bar-Shir, K. Yarlagadda, J. W. Bulte, P. B. Fisher, H. Wang, A. A. Gilad and M. G. Pomper, Magn Reson Med, 2015, 74, 544-549. X. Wang, Y. Wu, T. C. Soesbe, J. Yu, P. Zhao, G. E. Kiefer and A. D. Sherry, Angew Chem Int Ed Engl, 2015, 54, 8662-8664. J. N. Liu, W. Bu and J. Shi, Chem Rev, 2017, 117, 6160-6224. L. F. Vistain, M. W. Rotz, R. Rathore, A. T. Preslar and T. J. Meade, Chem Commun (Camb), 2016, 52, 160-163. M. W. Rotz, K. S. Culver, G. Parigi, K. W. MacRenaris, C. Luchinat, T. W. Odom and T. J. Meade, ACS Nano, 2015, 9, 3385-3396. N. Rammohan, R. J. Holbrook, M. W. Rotz, K. W. MacRenaris, A. T. Preslar, C. E. Carney, V. Reichova and T. J. Meade, Bioconjug Chem, 2017, 28, 153-160. R. J. Holbrook, N. Rammohan, M. W. Rotz, K. W. MacRenaris, A. T. Preslar and T. J. Meade, Nano Lett, 2016, 16, 3202-3209. F. J. Nicholls, M. W. Rotz, H. Ghuman, K. W. MacRenaris, T. J. Meade and M. Modo, Biomaterials, 2016, 77, 291-306. N. Rammohan, K. W. MacRenaris, L. K. Moore, G. Parigi, D. J. Mastarone, L. M. Manus, L. M. Lilley, A. T. Preslar, E. A. Waters, A. Filicko, C. Luchinat, D. Ho and T. J. Meade, Nano Lett, 2016, 16, 7551-7564. J. W. Zwanikken, P. Guo, C. A. Mirkin and M. Olvera de la Cruz, The Journal of Physical Chemistry C, 2011, 115, 16368-16373. H. Hinterwirth, S. Kappel, T. Waitz, T. Prohaska, W. Lindner and M. Lämmerhofer, ACS Nano, 2013, 7, 1129-1136. M. F. Ferreira, B. Mousavi, P. M. Ferreira, C. I. O. Martins, L. Helm, J. A. Martins and C. F. G. C. Geraldes, Dalton transactions, 2012, 41, 5472-5475.



49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.

R. J. Holbrook, N. Rammohan, M. W. Rotz, K. W. MacRenaris, A. T. Preslar and T. J. Meade, Nano Letters, 2016, 16, 3202-3209. L. c. Moriggi, C. Cannizzo, E. Dumas, C. d. R. Mayer, A. Ulianov and L. Helm, Journal of the American Chemical Society, 2009, 131, 10828-10829. J. M. Abad, M. Gass, A. Bleloch and D. J. Schiffrin, Journal of the American Chemical Society, 2009, 131, 10229-10236. S. D. Brown, P. Nativo, J.-A. Smith, D. Stirling, P. R. Edwards, B. Venugopal, D. J. Flint, J. A. Plumb, D. Graham and N. J. Wheate, Journal of the American Chemical Society, 2010, 132, 4678-4684. Y. Dong, S. Abaci, C. Shannon and M. J. Bozack, Langmuir, 2003, 19, 8922-8926. J. Park, S. Bhuniya, H. Lee, Y.-W. Noh, Y. T. Lim, J. H. Jung, K. S. Hong and J. S. Kim, Chemical Communications, 2012, 48, 3218-3220. J. A. Dougan, C. Karlsson, W. E. Smith and D. Graham, Nucleic Acids Research, 2007, 35, 36683675. M. Koufaki, C. Kiziridi, F. Nikoloudaki and M. N. Alexis, Bioorganic & Medicinal Chemistry Letters, 2007, 17, 4223-4227. J. A. Díaz, D. M. Grewer and J. M. Gibbs-Davis, Small, 2012, 8, 873-883. J.-Y. Chang, H. Wu, H. Chen, Y.-C. Ling and W. Tan, Chemical Communications, 2005, DOI: 10.1039/B414059A, 1092-1094. R. Karamanska, B. Mukhopadhyay, D. A. Russell and R. A. Field, Chemical Communications, 2005, DOI: 10.1039/B503843J, 3334-3336.


Page 14 of 18

Page 15 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dt-Gd(III)-SNA

3’ HS – TTT – TTT – TTT – TTT – TTT – Cy3-5’

5


4


a) Poly T Backfilled AuNP in 10% FBS Normalized Absorbance (AU)

1.0 0.8 0.6 0.4 0.2

500

550

600

650

0.8 0.6 0.4 0.2 0.0 450

wavelength (nm)

b)

500

550

600

650

1.0

time 0 1 day 7 days 14 days water control

0.8 0.6 0.4 0.2 0.0 450

wavelength (nm)


500

550

600

wavelength (nm)

10% FBS Supernatant Analysis

100

FBS Supernatant Analysis

Gd-DNA AuNP


100

Gd-DNA AuNP

80

Gd loss (%)

80

Gd loss (%)

0.0 450

Poly T Backfilled AuNP in FBS

1.0

Normalized Absorbance (AU)

Poly T Backfilled AuNP in PBS Normalized Absorbance (AU)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 18

60 40 20

60 40 20

0

0 0

100

200

300

400

0

100

time (hr)

200

time (hr)


300

400

650

Page 17 of 18

a

b 24 hr Cellular Uptake of Gd(III)-SNA Conjugates Backfilled poly dT SNAs

24 hr Cellular Uptake of Gd(III)-SNA Conjugates

Gd(III)-DNA SNAs

Backfilled poly dT SNAs

Gd(III)-DNA SNAs

0.5


0.5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.4 0.3 0.2 0.1

0.4 0.3 0.2 0.1 0.0

0.0 0

20

40

60

0

Gd(III) Dosing Concentration ( µM)


50

100

AuNP Dosing Concentration (nM)

150



Page 18 of 18

A Markedly Improved Synthetic Approach for the Preparation of

Recommend Documents