Letter www.acsami.org
Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
X‑ray-Mediated Release of Molecules and Engineered Proteins from Nanostructure Surfaces Mengqi Su,† Kathryn G. Guggenheim,‡ Jennifer Lien,† Justin B. Siegel,†,‡,§ and Ting Guo*,† †
Department of Chemistry, ‡Genome Center, and §Department of Biochemistry & Molecular Medicine, University of California, Davis, California 95616, United States
ACS Appl. Mater. Interfaces Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 09/14/18. For personal use only.
S Supporting Information *
ABSTRACT: Many applications call for initiation of chemical reactions with highly penetrating X-rays with nanometer precision and little damage to the surroundings, which is difficult to realize because of low interaction crosssections between hard X-rays and organic matters. Here, we demonstrate that a combination of computational protein design of single conjugation site green fluorescent proteins and nanomaterial engineering of silica-covered gold nanoparticles can enhance the release efficiencies of proteins from the surface of nanoparticles. The nanoparticles, to which the proteins are attached through DNA linkers, provide increased X-ray absorption without scavenging radicals, and single conjugation sites allow efficient release of proteins.
KEYWORDS: X-ray triggered release, engineered proteins, DNA strand breaks, gold nanoparticles, silica coating, surface functionalized nanoparticles, enhanced release, X-ray mediated reactions
T
the reference and silica-coated gold nanoparticles (AuNP@ SiO2) are used as the X-ray absorbing counterpart. Figure S1 shows TEM images of SiO2NPs and AuNP@SiO2. Three molecular reporters, 6-Carboxyfluorescein (6-FAM), cisplatin, and green fluorescent protein (GFP), which included the wildtype superfolder GFP and a novel computationally redesigned superfolder GFP, were attached to the surface of nanoparticles through 12 mer single-stranded DNA (ssDNA) molecules, which are susceptible to breakage through reactions with reactive oxygen species (ROS) such as hydroxyl radicals produced from electrons released from gold nanoparticles (thick red lines) and water (thin blue lines) upon X-ray irradiation, as shown in Figure 1B. Released reporters were detected using either mass spectrometry or fluorimetry. The use of large AuNPs can significantly increase the amount of radicals produced near the surface of nanoparticles, whereas the use of silica layer can effectively minimize scavenging of gold surface toward radicals. Figure 1C shows the theoretically predicted energy deposition enhancement using a recently developed package.7 An enhancement of 10.3 dose enhancement units (DEU) is found at the surface of gold nanoparticles (AuNPs) and the enhancement decreases to 4.1 DEU for 90 nm diameter gold nanoparticles coated with a layer of 15 nm (blue line) thick silica (AuNPs@SiO2). The enhancement is nearly 8.0 DEU when 1 nm silica layer is used (purple line).
he ability to initiate chemical and biological reactions with highly penetrating X-rays and nanometer spatial resolution has the potential to transform disease diagnostics and treatment, sensing, chemical synthesis, and lithography.1−6 Recent efforts have begun exploring the use of enhanced energy deposition near the surface of nanoparticles of heavy elements, such as gold nanoparticles, in order to initiate targeted chemical reactions through X-ray irradiation.7−10 However, current methods to release even small molecules require a large excess of X-ray exposure (∼100 Gy) to the point well beyond what is considered lethal and impractical for most applications of interest.11 Although release of molecules from nanomaterials have been reported recently,12,13 for macromolecules such as proteins, the dose to release could be higher due to the need to break multiple linkers. Therefore, novel approaches are needed to enhance the transformation of X-ray irradiation into chemical reactions. Here, we engineered and characterized proteins in nanochemical systems in which X-rays are used to achieve significant enhancement of the targeted chemical reactions. This outcome sets the stage for the conjugation and release of any species from nanostructures, and one example is releasing proteins of medicinal relevance that could fundamentally change how protein therapeutics are delivered and activated in biological systems. Figure 1A shows the overall design of the experiments used for characterizing a series of engineered nanosystems that enable enhancement of chemistry initiated through X-ray irradiation. Chemicals, syntheses, and procedures are described in the Supporting Information. Silica nanoparticles (SiO2NPs), which absorb X-rays slightly stronger than water, are used as © XXXX American Chemical Society
Received: August 1, 2018 Accepted: September 12, 2018
A
DOI: 10.1021/acsami.8b13117 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces
Figure 1. Nanochemical systems to support the detection of local enhancement. (A) Overall design of reporters that are conjugated to the surface of nanostructures and how enhancement is measured. SiO2NPs and AuNP@SiO2 are used. Reporters are conjugated to DNA that are attached to the surface of nanoparticles. Upon X-ray irradiation, the reporters are released from the nanoparticles and can be probed with mass spectrometry or fluorimetry. (B) shows electron emission (thick red and thin blue lines) and OH radical production as well as ligand release from AuNP@SiO2 (top panels) and SiO2NPs (bottom panels). (C) shows theoretical simulation results of energy deposition of electrons emitted from SiO2NPs (dashed line), AuNPs (red line), and AuNP@SiO2 with a 1 nm (purple) and 15 nm (blue line) thick silica layer.
Figure 2. (A) Experimental design using both wild-type (XN-GFP) and engineered (1N-GFP) proteins to demonstrate that undamaged fluorescent proteins can be released from nanostructures. (B) 1NGFP/XN-GFP emission and excitation fluorescence spectra, showing little or no change in the spectra. (C) describes the release of XNGFP and 1N-GFP from the surface of nanoparticles. (D) shows a percentage of XN-GFP (left bars) and 1N-GFP (right bars) release from three types of nanoparticles due to cleavage of single stranded DNA.
singly alkylated (Figure S5). Under equivalent conditions for 1N-GFP, as shown in Figures S3 and S6 and S7, the primary product is a protein species with one chemically conjugated alkyne, as designed (Figure S7). The labeled XN and 1N-GFP product were then covalently linked to either AuNP@SiO2 or SiO2NPs. Conjugation of GFP molecules to the surface of AuNP@SiO2 is shown in Figure 2A, which include three steps, as explained in the Supporting Information. Upon DNA strand breaks by reacting with ROS, XN- and 1N-GFP reporters are released, as indicated in Figure 2C. A higher dose should be needed to release XN-GFP due to a higher number of DNA linkers between the GFP and the surface of nanoparticles. The release results are shown in Figure 2D and Table 1, and percentage release for 1N-GFP increases 8-fold compared to
The design and mechanisms used to test the release of GFP from nanochemical systems are studied in this work and shown in Figure 2A. Here, two variants of GFP were tested, the wildtype superfolder GFP (termed XN-GFP here, PDB accession number 2B3P)14 that has multiple conjugation points and a reengineered variant that was designed to enable a single-point chemical conjugation between the nanoparticle and protein molecule (termed 1N-GFP). Specifically, chemical conjugation of the nanoparticle to GFP through amines in the protein was utilized. To design 1N-GFP, the Rosetta Molecular Modeling Suite15 was used to evaluate the energetics of mutating each lysine in the protein to arginine, and predicted 19 of the 20 could be mutated without compromising the protein structure (Table S1 and Figure S2). A synthetic gene was constructed which encoded 1N-GFP which has only one lysine, predicted to be buried and critical for structure, and the N-terminal amine of the protein available for NHS-ester conjugation chemistry.16 Sequences of 1N- and XN-GFP are shown in Table S2. Proteins were then produced in E. coli, purified, and experimentally characterized. The excitation and emission spectra depict (Figure 2B) that engineering and chemically altering XN-GFP has little or no effect on fluorescence intensity and pattern. XN-GFP, as shown in Figures S3−S5, is observed to have a distribution of 0, 1, 2, 3, and 4 chemically conjugated alkynes, with less than 30% of the material being
Table 1. Percentage Cleavage and Enhancement Values for Both Wild-Type and Engineered GFP Attached to the Ends of DNA Strands Anchored at the Surface of Two Types of Nanoparticlesa protein type
SiO2NPs (% cleavage)
AuNP@SiO2 (% cleavage)
1N-GFP 0.12 ± 0.02 4.33 ± 0.44 XN-GFP 0.015 ± 0.004 2.03 ± 0.25 XN-GFP (SiO2NPs)/1N-GFP (AuNP@SiO2)
enhancement (DEU) 36 ± 6 135 ± 40 288
All GFP irradiation was performed in 0.01 wt % (∼81 μM) Tween20 and 10 mM PBS. No reduction to GFP fluorescence was observed.
a
B
DOI: 10.1021/acsami.8b13117 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces
probability of having multiple OH radicals in each unit volume as a function of average OH radicals per unit volume is shown in Figure 3B. The four marked points show the cases compatible to experimental conditions. For SiO2NPs, percentages of units having 1 and 2 radicals are 6% (blue triangle) and 0.1% (blue circle) for 0.05 average OH radicals per unit of a 13 nm radius cylinder. In contrast, the probabilities of units having 1 or 2 radicals increase to 63% (orange triangle) or 26% (orange circle) for an average of 0.8 OH radicals per unit of a 25 nm diameter cylinder for AuNP@SiO2. This result clearly explains why 1N-GFP with a single DNA linker connecting to AuNP@SiO2 is more readily released, and requires only one OH radical, than XN-GFP connected to SiO2NPs through multiple DNA linkers, which requires multiple OH radicals. The steric hindrance of more rigidly positioned XN-GFP on the surface of SiO2NPs blocking the reaction paths of OH radicals produced further away from the nanoparticle surface is also modeled. The average solid angle subtended by the DNA strand for each radical is shown in Figure 3C. Finally, the results shown in Figure 3B, C can be combined to obtain the cleavage efficiency in %cleavage, which should be proportional to the multiplication of the average solid angle and probability of having the needed number of OH radicals in the reaction unit volume. The trend of the calculated enhancements for all four cases shown in Figure 3D is consistent with the trend of the experimentally determined enhancements shown in Table 1. To further confirm the observed local enhancement for the engineered nanoparticles, 6-FAM and cisplatin reporters were used to quantitatively measure X-ray mediated release of small molecules. Figure 4A shows the synthetic route. Figure 4B (bottom panel) shows the results of measurements using matrix assisted laser desorption ionization (MALDI) mass spectrometry to detect 6-FAM DNA ligands. The percentage of 6-FAM released from the nanostructures and into the supernatant after X-ray irradiation is shown in Figure 4C, obtained by measuring the fluorescence signal from released 6FAM in supernatants. Unlike irradiation of GFP with Tween20 with X-rays in which no damage of GFP was observed, irradiation of 6-FAM caused ca. 55% damage using the same dose. The release efficiency of 6-FAM after damage correction is 0.33 ± 0.01% from SiO2NPs and 3.15 ± 0.05% from AuNP@SiO2, giving rise to a 9.7 DEU enhancement. These results are the first direct measurement of local or type 2 physical enhancement without using nanoreactors such as calcium phosphate enclosed liposomes (CaPELs).18 For the cisplatin-DNA conjugation approach, which is described in the Supporting Information, an inductive-coupled plasma-mass spectrometry (ICP-MS) method was developed to detect platinum released from nanostructures. Although cisplatin has been conjugated to the surface of gold nanoparticle before, this was the first case where cisplatin mass spectrometry was used to measure the enhancement.19 The overall design of the experiment is shown in Figure 4A as well. The MALDI data is given in Figure 4B (top panel); it shows that for each 12-mer DNA, there were on average 1.6 cisplatin molecules based on measurements using mass spectrometry. Figure 4C shows the results of ICP-MS measurements. For cisplatin release, the efficiencies were 0.047 ± 0.017% for SiO2NPs and 0.500 ± 0.014% for AuNPs@SiO2, which translates to an enhancement of 10.6 DEU. No correction for cisplatin damage is needed because MALDI only detects the addition of cisplatin compounds to
XN-GFP for SiO2NPs and is doubled for AuNP@SiO2 (red bars). GFP release in the form of 1N-GFP and XN-GFP are 2.16 ± 0.15% and 1.04 ± 0.08%, respectively, from the surface of AuNPs under X-ray irradiation. When SiO2NPs were used, the percentage was significantly lower (gray bars). GFP emission spectra, shown in Figure S8, remained nearly constant after ca. 30 Cy irradiation, suggesting that GFP molecules were not damaged. Although proteins react with ROS at high rates,9 the low concentrations of ROS made such damage (less than one reaction per protein) undetectable in this work. Release efficiency from AuNP@SiO2 could potentially be four times higher if (1) the thickness of SiO2 coating is reduced from 15 to 1 nm and (2) DNA strand breaks are chemically enhanced, both are possible.4,7 This means that the X-ray dose required for release could potentially be further reduced to less than 10 Gy (∼7.5 Gy) as the whole system is further optimized. For the release of XN-GFP, adding a gold core to the silica nanoparticles increased the release efficiency by 135-fold, to 2.03%. For the release of engineered 1N-GFP, this improvement was 36-fold, to 4.33%. The absolute efficiencies from AuNP@SiO2 are higher than release from AuNPs, because the silica coating lessens scavenging of the gold surface even though this coating also reduces local energy deposition enhancement as well as chemical enhancement. When the release of engineered GFP from AuNP@SiO2 is compared with wild-type GFP from SiO2NPs, the combined nanomaterial engineering and protein engineering enables a 288-fold increase, as shown in Table 1, a result fully demonstrating the benefits of using these new designs implemented in this work. To better understand the mechanism of enhancement, we performed a theoretical simulation that focused on the geometric possibility of ROS interactions with and cleavage of ssDNA in these different systems. This mechanism is different than electron−DNA or UV−DNA interactions, which predominantly occur through direct photoexcitation of bases.17 The simulation geometry is defined in Figure 3A. The
Figure 3. Illustration of the overall enhancement mechanism. (A) How a GFP is positioned in a three-dimensional cylinder volume unit. (B) Probabilities of having 1, 2, and 4 radicals in the same unit described in A as a function of average number of OH radicals in the unit. (C) Results of calculated average solid angles of collisions between an OH radical and the DNA strand on the surface of SiO2NPs and AuNP@SiO2. (D) Predicted % cleavage in each case, which are the multiplication of results shown in Figure 3B, C. 2NGFP is used here. C
DOI: 10.1021/acsami.8b13117 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding
This work is supported in part by the UC Cancer Research Coordinating Committee (CRR-15-380513) and the National Science Foundation (CHE-1307529). Notes
The authors declare no competing financial interest.
■
Figure 4. Release of 6-FAM or cisplatin from AuNP@SiO2 and SiO2 NPs. (A) Construct of the nanochemical system in which 6-FAM or cisplatin is attached to the surface of gold nanoparticles through DNA ligands. (B) MALDI results of cisplatin conjugated 12-mer DNA (top panel). 6-FAM-DNA was also measured using MALDI for comparison purposes (bottom panel). (C) Raw signals of 6-FAM (fluorescence, bars on the right) released from SiO2NPs and AuNP@ SiO2. The experimental ICP-MS results (bars on the left) of using cisplatin to determine the enhancement are also shown.
DNA molecules and no modification to the complex was found. These results demonstrate that it is possible to utilize engineered nanomaterials to amplify the release of small fluorescent molecules and especially engineered proteins after exposure to low doses of X-rays. Due to the penetrating nature of X-rays and the ability to achieve three-dimensional spatial and temporal precision, the engineered system opens the possibility of carrying out targeted chemical reactions with nanoscale precision in an unprecedented manner. Of particular interest is the potential this system has for controlling chemistry within a biological system. Through the demonstrated release of both small molecules and proteins, one can imagine imminent applications toward highly targeted prodrug delivery and a subsequent X-ray-mediated activation system.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b13117. Experimental procedures, chemicals, methods of synthesis, and characterization results (PDF)
■
REFERENCES
(1) Cormode, D. P.; Naha, P. C.; Fayad, Z. A. Nanoparticle contrast agents for computed tomography: a focus on micelles. Contrast Media Mol. Imaging 2014, 9, 37−52. (2) Hainfeld, J. F.; Slatkin, D. N.; Smilowitz, H. M. The use of gold nanoparticles to enhance radiotherapy in mice. Phys. Med. Biol. 2004, 49, N309−N315. (3) Foley, E.; Carter, J.; Shan, F.; Guo, T. Enhanced relaxation of nanoparticle-bound supercoiled DNA in X-ray radiation. Chem. Commun. 2005, 3192−3194. (4) Cheng, N. N.; Starkewolf, Z.; Davidson, A. R.; Sharmah, A.; Lee, C.; Lien, J.; Guo, T. Chemical Enhancement by Nanomaterials under X-ray Irradiation. J. Am. Chem. Soc. 2012, 134, 1950−1953. (5) Davidson, R. A.; Guo, T. An Example of X-ray Nanochemistry: SERS Investigation of Polymerization Enhanced by Nanostructures under X-ray Irradiation. J. Phys. Chem. Lett. 2012, 3, 3271−3275. (6) Kim, T.; Zaera, F. X-ray-Initiated Metal-Promoted Thin Film Growth. J. Phys. Chem. C 2012, 116, 8594−8600. (7) Lee, C.; Cheng, N. N.; Davidson, R. A.; Guo, T. Geometry Enhancement of Nanoscale Energy Deposition by X-rays. J. Phys. Chem. C 2012, 116, 11292−11297. (8) Casta, R.; Champeaux, J.P.; Sence, M.; Moretto-Capelle, P.; Cafarelli, P.; Amsellem, A.; Sicard-Roselli, C. Electronic emission of radio-sensitizing gold nanoparticles under X-ray irradiation: experiment and simulations. J. Nanopart. Res. 2014, 16, 2348. (9) Farhataziz; Rodgers, M .A. J. Radiation Chemistry: Principles and Applications; VCH Publishers: New York, 1987; p 527. (10) Guo, T. X-ray nanochemistry: Concepts and Development. Nanostructure Science and Technology; Springer: New York, 2018; Vol. XXI, p 513. (11) Starkewolf, Z. B.; Miyachi, L.; Wong, J.; Guo, T. X-ray triggered release of doxorubicin from nanoparticle drug carriers for cancer therapy. Chem. Commun. 2013, 49, 2545−2547. (12) Zhou, Z. J.; Chan, A.; Wang, Z. T.; Huang, X. L.; Yu, G. C.; Jacobson, O.; Wang, S.; Liu, Y. J.; Shan, L. L.; Dai, Y. L.; Shen, Z. Y.; Lin, L. S.; Chen, W.; Chen, X. Y. Synchronous Chemoradiation Nanovesicles by X-Ray Triggered Cascade of Drug Release. Angew. Chem., Int. Ed. 2018, 57, 8463−8467. (13) Liu, F.; Lou, J. Z.; Hristov, D. X-Ray responsive nanoparticles with triggered release of nitrite, a precursor of reactive nitrogen species, for enhanced cancer radiosensitization. Nanoscale 2017, 9, 14627−14634. (14) Pedelacq, J. D.; Cabantous, S.; Tran, T.; Terwilliger, T. C.; Waldo, G. S. Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 2006, 24, 79−88. (15) Leaver-Fay, A.; Tyka, M.; Lewis, S.M.; Lange, O.F.; Thompson, J.; Jacak, R.; Kaufman, K.; Renfrew, P.D.; Smith, C.A.; Sheffler, W.; Davis, I.W.; Cooper, S.; Treuille, A.; Mandell, D.J.; Richter, F.; Ban, Y.E.A.; Fleishman, S.J.; Corn, J.E.; Kim, D.E.; Lyskov, S.; Berrondo, M.; Mentzer, S.; Popovic, Z.; Havranek, J.J.; Karanicolas, J.; Das, R.; Meiler, J.; Kortemme, T.; Gray, J.J.; Kuhlman, B.; Baker, D.; Bradley, P. Rosetta3: An Object-Oriented Software Suite for the Simulation and Design of Macromolecules. Methods in Enzymol. 2011, 487, 545− 574. (16) Stephanopoulos, N.; Francis, M. B. Choosing an effective protein bioconjugation strategy. Nat. Chem. Biol. 2011, 7, 876−884.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Ting Guo: 0000-0002-6700-0967 D
DOI: 10.1021/acsami.8b13117 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces (17) Greenberg, M. M., ed. Radical and Radical Ion Reactivity in Nucleic Acid Chemistry; Rokita, S.E., Ed.; Wiley Series on Reactive Intermediates in Chemistry and Biology; John Wiley & Sons: Hoboken, NJ, 2009. (18) Sharmah, A.; Yao, Z.; Lu, L.; Guo, T. X-ray-Induced Energy Transfer between Nanomaterials under X-ray Irradiation. J. Phys. Chem. C 2016, 120, 3054−3060. (19) Setua, S.; Ouberai, M.; Piccirillo, S. G.; Watts, C.; Welland, M. Cisplatin-tethered gold nanospheres for multimodal chemo-radiotherapy of glioblastoma. Nanoscale 2014, 6, 10865−10873.
E
DOI: 10.1021/acsami.8b13117 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
