Nucleic Acid Nanocapsules for Enzyme-Triggered Drug Release

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Nucleic Acid Nanocapsules for Enzyme-Triggered Drug Release Joseph K. Awino, Saketh Gudipati, Alyssa K. Hartmann, Joshua J. Santiana, Dominic F. Cairns-Gibson, Nicole Gomez, and Jessica L. Rouge* Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, United States S Supporting Information *

scaffold for further RNA and DNA functionalization. Although there are numerous soft material based approaches to drug encapsulation,17 the specific challenge for this study was to develop a material that can be rapidly functionalized with nucleic acids and rapidly release a drug cargo. This way the particle could impart enhanced uptake due to the properties of the SNA, coupled with gene knockdown potential and small molecule drug delivery in a single construct. Herein we describe a straightforward assembly strategy of a nanoparticle formulation that is suitable for both the attachment of a catalytic oligonucleotide at its surface and the encapsulation of a small molecule drug in its interior. Through this approach both the interior and exterior of the construct can be utilized for maximal therapeutic effect. This construct is referred to here as a nucleic acid nanocapsule (NAN) and is designed to degrade in the presence of endogenous cellular enzymes. The NAN’s core is synthesized using a two-step approach combining self-assembly and a surface cross-linking step inspired by surfactants developed for chemically triggered release.18 The major difference in the NAN’s core design is in the cross-linking step which can be used to trigger an enzymatic disassembly step, important for biochemically controlled drug release. First, an alkyne-terminated surfactant 1 was synthesized (details in SI) and self-assembled in water. After assembly the particles are covalently cross-linked to hold the micelle-like structure intact. The cross-linker 2 is functionalized with azido groups on either end to facilitate cross-linking to the alkyne head groups presented by the surfactant.19 This cross-linker is incorporated into the particles design in order to stabilize the nanocapsule’s core and recruit enzymes for initiating its degradation. Esterases are enzymes that can rapidly recognize and cleave ester bonds and have been shown to be effective targets for catalyzing drug release from nanomaterials.20 In addition to stabilizing the particle with ester linkages the NANs gain the potential to release hydrophobic small molecules from their core in response to enzymes (Figure 1). Esterases were also specifically chosen as the biochemical trigger for degrading the nanocapsules as these enzymes are known to be concentrated inside cellular endosomes.21,22 It is well established that SNA-like structures are taken up through scavenger-receptor mediated endocytosis and that their ultimate fate is within endosomes.23 As many drugs and therapeutic antisense oligonucleotides need to reach the cytosol to exert their therapeutic effect,24,25 it was of interest to release

ABSTRACT: Herein we describe a nucleic acid functionalized nanocapsule in which nucleic acid ligands are assembled and disassembled in the presence of enzymes. The particles are fully degradable in response to esterases due to an embedded ester cross-linker in the particle’s core. During synthesis the nanocapsules can be loaded with hydrophobic small molecules and post self-assembly undergo covalent cross-linking using copper catalyzed click chemistry. They can then be functionalized with thiolated DNA through stepwise thiolyne chemistry using UV light irradiation. Additionally, the capsule is compatible with enzyme mediated functionalization of a therapeutic mRNA-cleaving DNAzyme at the particle’s surface. The resulting particle is highly stable, monodisperse in size, and maximizes the therapeutic potential of both the particles interior and exterior.

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ucleic acid-based therapeutics have become increasingly important drug candidates in recent years thanks to the advent of nanoparticle drug carriers. Such nucleic acids, including siRNA,1−3 antisense DNA4,5 and catalytic nucleic acids6,7 have been shown to be effective tools for initiating intracellular gene regulation. However, these molecules suffer in their overall efficacy due to their inherent chemical instability.5 Recent studies have shown that the tight packing of nucleic acids at the surface of a nanoparticle can result in advantageous cellular delivery properties that the nucleic acid sequence alone cannot achieve.8,9 Such structures, referred to as spherical nucleic acids (SNAs) provide desirable delivery properties including increased cellular uptake through endocytosis and the prolonged half-life of nucleic acids.10,11 The later property is particularly important for the delivery of nucleic acids that rely on their folded structure to impart therapeutic effects including DNAzymes, ribozymes and aptamers.12−14 Using such an SNA configuration on a colloidal gold nanoparticle scaffold, it was recently shown that a functional ribozyme could be successfully delivered into cells for regulating gene expression.15 However, as these structures were attached to inorganic nanoparticles (NPs), much of the particle core could not contribute to the overall therapeutic function other than to provide a scaffold on which to build the SNA configuration. In light of this underutilized aspect of the SNA, studies are now focusing on utilizing the core composition of an SNA as a drug carrier.16 For the current study, it was of interest to design a chemical strategy for assembling an SNA-like structure at the surface of a nanomaterial that could be utilized as a drug carrier and as a © XXXX American Chemical Society

Received: December 20, 2016

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DOI: 10.1021/jacs.6b13087 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Figure 1. Stepwise assembly of nucleic acid nanocapsules. The trialkylmodified surfactant 1 shown at top left is placed in water and quickly self-assembles into a micelle structure presenting alkynes at its surface. An esterified diazido cross-linker 2 is used to stabilize the structure using copper I catalyzed click chemistry. Remaining alkynes are used as a point of attachment for thiolated DNA molecules through a photodriven cross-linking step. After assembly of the NAN structure it can be further functionalized using an enzyme-mediated assembly approach with T4 DNA ligase to introduce DNAzymes to the NAN’s surface.

Figure 3. Characterization of NANs pre and post DNA assembly. (A) Zeta potential measurements of the nanocapsules pre and post functionalization with DNA. (B) 1% agarose gel showing movement of fluorescent NANs loaded with rhodamine B through an electric field post DNA functionalization (1, cross-linked micelle prior to DNA attachment; 2, NAN, post DNA attachment).

the contents of the NAN once inside the cell in order to increase the likelihood its contents would make it to the cytosol. Through careful control over the stoichiometry of the diazido cross-linker relative to the total number of alkynes presented at the particle surface (1:1.2 respectively), enough alkynes could be left unreacted to allow for attachment of a thiolated nucleic acid in the second assembly step. Using this approach thiolated DNA was attached to the surface of the nanocapsule using UV irradiation (365 nm) and a water-soluble photoinitiator traditionally used in polymerization reactions.26,27 The resulting NAN is monitored during each step of assembly through a series of characterization techniques including dynamic light scattering (DLS), transmission electron microscopy (TEM) and zeta potential measurements (Figure 2).

nm as would be expected for the attachment of a 22mer DNA strand (Figure 2B). Additionally, post DNA functionalization, the once highly positively charged nanocapsule could now migrate through a 1% agarose gel under an electric field, indicative of the presence of the negatively charged DNA strands at the particles surface (Figure 3B). After assembling the NANs it was of interest to test their ability to release a small molecule from its interior. To determine this, NANs were synthesized in the presence of rhodamine B dye. The effective loading of the particles was optimized at 2.5% loading by concentration although loading as high as 10% was possible (Figure S4). The resulting particles were subjected to treatment with porcine liver esterase and monitored using fluorescence spectroscopy. The NANs containing ester linkages were successfully cleaved as indicated by an increase in the samples fluorescence over time (Figure 4A). As a control, a nanocapsule cross-linked with non-ester linkages was prepared and treated with esterase and release of the dye was not observed (Figure 4B). The lack of dye release indicates that the stability of the particle is gated by the enzymatic environment of the nanocapsule. The NANs were also evaluated for their cell toxicity effects and shown to be nontoxic up to micromolar concentrations (Figure S7). Once it was established in vitro that the particles were esterase-responsive it was of interest to incubate cells with the NANs to see if the SNA-like construct could result in cellular uptake without transfection agents, a feature exhibited by SNA configurations on nanoparticle surfaces. Confocal studies showed that the particles were indeed readily taken up by cells in a fashion similar to that seen with traditional SNA-like structures (see Figure 5C−F) and are shown to colocalize within regions of the cell that indicate endocytosis as the main mechanism of uptake. Knowing that the NANs could enter cells and are biocompatible, their ability to effectively deliver a small molecule drug was then evaluated. The NANs were loaded with camptothecin, an apoptosis inducing drug28,29 and incubated with HeLa cells for 4 h at 37 °C. Cell proliferation

Figure 2. Characterization of NANs pre and post DNA assembly. (a) Dynamic light scattering measurements of particles prior to and after DNA conjugation, cross-linked micelle (CM) and NAN. (A) TEM micrograph showing the average size of uranyl acetate stained NANs.

The cross-linked micelle (CM) prior to DNA conjugation exhibited a uniform size distribution (20 ± 3 nm) and positive charge (+50 ± 4 mV). Such a positive surface charge was expected due to the presence of the tertiary amines presented by the alkyne functionalized headgroup. Post DNA attachment, the particles exhibited a dramatic shift in surface charge to −40 mV (Figure 3A). The particle size also increased from 20 to 34 B

DOI: 10.1021/jacs.6b13087 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Figure 4. Enzyme-mediated release of NANs internal cargo. (A) Total emission in a sample of NANs cross-linked with compound 2 containing rhodamine B, post treatment with esterase over time in buffer. (B) Representative plots of ester-linked NANs versus nonester-linked NANs (made with non-esterified crosslinker (Figure S2) versus time after 2 h of treatment with esterase. (C,D) Evaluation of cell toxicity in HeLa cells in the presence of NANs loaded with camptothecin. Results indicate a dose dependent decrease in cell viability post 24 h incubation with ester-linked NANs (red bars) in (C), and a limited effect from NANs linked by a non-esterified linker (blue bars) in (D). Concentrations indicate total camptothecin loaded within NANs. Control is 50 μM free drug. These results indicate that the release of the apoptotic drug is dependent upon the presence of both the esterified cross-linker and the esterase.

Figure 5. Cleavage of mRNA truncates using DNAzyme-functionalized NANs and cellular uptake. (A) Polyacrylamide gel electrophoresis showing the cleavage of RNA induced by DNAzymefunctionalized NANs after 4 h at 37 °C: lane 1, truncated mRNA free in solution; lane 2, post incubation with DNAzyme-NANs; lane 3, mutated DNAzyme-NANs without salts; lane 4, mutated DNAzymeNANs with salts; and lane 5, DNAzyme off particle, showing complete cleavage of the truncated mRNA target. Mutated DNAzyme details in SI. Panel B shows the DNAzyme-NAN is able to cleave a fluorescently labeled mRNA truncate (Table 1, SI for details) that is quenched prior to cleavage but becomes fluorescent post cleavage. In the presence of the DNAzyme-functionalized NANs, the mRNA truncate (10 nM) is rapidly cleaved as shown above. DNAzyme concentrations used in the ligation reaction can result in multiple DNAzymes per NAN. Fluorescence traces are shown to indicate activity versus lack of activity. Free DNAzyme (500 nM) along with NANs functionalized with mutated DNAzyme-NANs (both at 100 nM) are shown as positive and negative controls for activity, respectively. (C−F) Cells treated with 1 μM rhodamine B loaded NANs: (C) green emission channel indicating the location of the rhodamine B NANs in cells; (D) Lysotracker Red staining of lysosomes and endosomes within cells; (E) co-localization of NANs and lysosomes showing the NANs enter the cells through endocytosis; (F) brightfield image and overlay. Scale bar is 25 μm.

studies showed that cells treated with camptothecin-loaded NANs (50 μM drug) effectively limited the growth of cells by 50% relative to untreated cells. Non-ester cross-linked NANs loaded with 50 μM drug had minimal effect on cell growth (Figure 4D). A central goal in designing the NAN was to make a material that could serve as a fully degradable scaffold for therapeutic nucleic acids with the potential to release a drug from its core. This functional aspect of the NAN construct was evaluated through the attachment of a DNAzyme that requires its folded structure to function in the cleavage of mRNA. The DNAzyme hgd40 was specifically chosen as a test oligonucleotide sequence as it has recently been shown to rapidly cleave mRNA encoding an important transcription factor (GATA-3) involved in inflammation pathways30,31 In order to design this construct the DNAzyme was first enzymatically assembled onto the NANs surface using a recently developed enzyme ligation approach compatible with SNA like structures.32 The DNAzyme-functionalized NANs were first incubated with an mRNA truncate of GATA-3 and then evaluated for evidence of mRNA cleavage in vitro. The DNAzyme-functionalized NANs were shown to be effective at cleaving a truncated GATA-3 mRNA sequence at 37 °C after 4 h as indicated by polyacrylamide gel electrophoresis (Figure 5A). To probe the relative amount of cleavage of the mRNA truncate in the presence of the DNAzyme-functionalized NAN, a modified mRNA truncate containing a dye (FAM) and a dye quencher (BHQ-1) was monitored using fluorescence spectroscopy. In the presence of 100 nM DNAzyme-NANs, 10 nM mRNA truncate is fully cleaved within the first hour of incubation as indicated by the increase in fluorescence observed (Figure 5B).

These results indicate that the particle surface can be easily modified using enzymes with functional, therapeutic nucleic acids. In summary, we have shown a straightforward assembly strategy for a monodisperse drug carrier that lends itself to both functionalization with nucleic acid ligands using enzyme mediated attachment and that whose degradation can be triggered using a second enzyme, esterase. The resulting construct can be loaded with hydrophobic small molecules including dyes and drugs for efficient enzyme-triggered release into cells. Such a formulation has the potential to be tailored to respond to a variety of different enzymes through the systematic synthesis of different chemically unique crosslinkers. Through tailoring the reactivity of the cross-linkers the NAN formulation can be designed to trigger its release in a C

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(14) Padlan, C. S.; Malashkevich, V. N.; Almo, S. C.; Levy, M.; Brenowitz, M.; Girvin, M. E. RNA 2014, 20, 447. (15) Rouge, J. L.; Sita, T. M.; Hao, L.; Mirkin, C. A.; et al. J. Am. Chem. Soc. 2015, 137, 10528. (16) Tan, X.; Lu, X.; Jia, F.; Liu, X.; Sun, Y.; Logan, J. K.; Zhang, K. J. Am. Chem. Soc. 2016, 138, 10834. (17) Sun, W.; Jiang, T.; Lu, Y.; Reiff, M.; Mo, R.; Gu, Z. J. Am. Chem. Soc. 2014, 136, 14722. (18) Zhang, S.; Zhao, Y. J. Am. Chem. Soc. 2010, 132, 10642. (19) Chen, C.; Li, J.; Ji, G.; Zhu, D.; Zheng, Q. Synth. Commun. 1998, 28, 3097. (20) Oh, S. S.; Lee, B. F.; Leibfarth, F. A.; Eisenstein, M.; Robb, M. J.; Lynd, N. A.; Hawker, C. A.; Soh, H. T. J. Am. Chem. Soc. 2014, 136, 15010. (21) Runquist, E. A.; Havel, R. J. J. Biol. Chem. 1991, 266, 22557. (22) Brockman, S. A.; Murphy, R. F. Pharmaceutical Biotechnology; Springer: New York, 1993; p 51. (23) Wu, X. A.; Choi, C. H. J.; Zhang, C.; Hao, L.; Mirkin, C. A. J. Am. Chem. Soc. 2014, 136, 7726. (24) Vasir, J. K.; Labhasetwar, V. Adv. Drug Delivery Rev. 2007, 59, 718. (25) Zeng, Y.; Cullen, B. R. RNA 2002, 8, 855. (26) Lowe, A. B. Polymer 2014, 55, 5517. (27) Lowe, A. B.; Hoyle, C. E.; Bowman, C. N. J. Mater. Chem. 2010, 20, 4745. (28) Zeng, C. W.; Zhang, X. J.; Lin, K. Y.; Ye, H.; Feng, S. Y.; Zhang, H.; Chen, Y. Q. Mol. Pharmacol. 2012, 81, 578. (29) Morris, E. J.; Geller, H. M. J. Cell Biol. 1996, 134, 757. (30) Sel, S.; Wegmann, M.; Dicke, T.; Sel, S.; Henke, W.; Yildirim, A. O.; Renz, H.; Garn, H. J. Allergy Clin. Immunol. 2008, 121, 910. (31) Krug, N.; Hohlfeld, J. M.; Kirsten, A.-M.; Kornmann, O.; Beeh, K. M.; Kappeler, D.; Korn, S.; Ignatenko, S.; Timmer, W.; Rogon, C.; Zeitvogel, J.; Zhang, N.; Bille, J.; Homburg, U.; Turowska, A.; Bachert, C.; Werfel, T.; Buhl, R.; Renz, J.; Garn, H.; Renz, H. N. Engl. J. Med. 2015, 372, 1987. (32) Rouge, J. L.; Hao, L.; Wu, X. A.; Briley, W. E.; Mirkin, C. A. ACS Nano 2014, 8, 8837.

more biochemically specific fashion, depending on its cellular environment. Through the enzyme-triggered recognition step the NAN construct has the potential to diminish off target effects that often occur during uptake of nanoparticle-based drug delivery systems and ensure a better route to uptake and efficacy for both small molecule drugs and therapeutic nucleic acids alike.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.6b13087. Experimental details, along with all nucleic acid sequences, synthetic strategies, and assay conditions (PDF)

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AUTHOR INFORMATION

Corresponding Author

* [email protected] ORCID

Joseph K. Awino: 0000-0003-3710-3014 Saketh Gudipati: 0000-0002-7886-1260 Jessica L. Rouge: 0000-0002-6051-3735 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This material is based upon work supported by the University of Connecticut. The authors thank Dr. Binglin Sui for help with the cell toxicity experiments.

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REFERENCES

(1) Judge, A. D.; Bola, G.; Lee, A. C. H.; MacLachlan, I. Mol. Ther. 2006, 13, 494. (2) Zimmermann, T. S.; Lee, A. C.; Akinc, A.; Bramlage, B.; Bumcrot, D.; Fedoruk, M. N.; Harborth, J.; Heyes, J. A.; Jeffs, L. B.; John, M.; Judge, A. D.; Lam, K.; McClintock, K.; Nechev, L. V.; Palmer, L. R.; Racie, T.; Rohl, I.; Seiffert, S.; Shanmugam, S.; Sood, V.; Soutschek, J.; Toudjarska, I.; Wheat, A. J.; Yaworski, E.; Zedalis, W.; Koteliansky, V.; Manoharan, M.; Vornlocher, H.-P.; MacLachlan, I. Nature 2006, 441, 111. (3) Xie, F. Y.; Woodle, M. C.; Lu, P. Y. Drug Discovery Today 2006, 11, 67. (4) Uhlmann, E.; Peyman, A. Chem. Rev. 1990, 90, 543. (5) Geary, R. S.; Norris, D.; Yu, R.; Bennett, F. Adv. Drug Delivery Rev. 2015, 87, 46. (6) Tedeschi, L.; Lande, C.; Cecchettini, A.; Citti, L. Drug Discovery Today 2009, 14, 776. (7) Wang, T. H.; Li, W. T.; Yu, S. H.; Au, L. C. Oligonucleotides 2008, 18, 295. (8) Cutler, J. I.; Auyeung, E.; Mirkin, C. A. J. Am. Chem. Soc. 2012, 134, 1376. (9) Giljohann, D. A.; Seferos, D. S.; Prigodich, A. E.; Patel, P. C.; Mirkin, C. A. J. Am. Chem. Soc. 2009, 131, 2072. (10) Patel, P. C.; Hao, L.; Au Yeung, W. S.; Mirkin, C. A. Mol. Pharmaceutics 2011, 8, 1285. (11) Narayan, S. P.; Choi, C. H. J.; Hao, L.; Calabrese, C. M.; Auyeung, E.; Zhang, C.; Goor, O. G. M.; Mirkin, C. A. Small 2015, 11, 4173. (12) Kim, H.-K.; Rasnik, I.; Liu, J.; Ha, T.; Lu, Y. Nat. Chem. Biol. 2007, 3, 763. (13) Shu, D.; Khisamutdinov, E. F.; Zhang, L.; Guo, P. Nucleic Acids Res. 2014, 42, e10. D

DOI: 10.1021/jacs.6b13087 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX