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Feb 26, 2016 - aluminum hydroxide (Mg/Al LDH),3,4,17,21−29 which can be described by the formula ... negatively charged DNA molecules, we studied the surface charge of the LDH ... “flat” on the NP surfaces, each DNA nucleotide will create a .... molecules approach LDH surfaces, the phosphate oxygen may initiate a ...

Article pubs.acs.org/Langmuir

Chemisorption Mechanism of DNA on Mg/Fe Layered Double Hydroxide Nanoparticles: Insights into Engineering Effective SiRNA Delivery Systems Mingsheng Lu,† Zhi Shan,‡ Kori Andrea,‡ Bruce MacDonald,‡ Stefanie Beale,§ Dennis E. Curry,‡,∥ Li Wang,⊥ Shujun Wang,# Ken D. Oakes,‡,∇ Craig Bennett,§ Wenhui Wu,*,† and Xu Zhang*,‡ †

College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, People’s Republic of China Verschuren Centre for Sustainability in Energy and the Environment, and ∇Department of Biology, Cape Breton University, 1250 Grand Lake Road, Sydney, Nova Scotia B1P 6L2, Canada § Department of Physics, Acadia University, Wolfville, Nova Scotia B4P 2R6, Canada ∥ Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada ⊥ College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, Zhejiang 310018, People’s Republic of China # Jiangsu Marine Resources Development Research Institute, Lianyungang, Jiangsu 222001, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: Layered double hydroxide nanoparticles (LDH NPs) have attracted interest as an effective gene delivery vehicle in biomedicine. Recent advances in clinic trials have demonstrated the efficacy of Mg/Fe LDHs for hyperphosphatemia treatment, but their feasibility for gene delivery has not been systematically evaluated. As a starting point, we aimed to study the interaction between oligo-DNA and Mg/Fe LDH NPs. Our investigation revealed the chemisorption mechanism of DNA on Mg/Fe LDH surfaces, wherein the phosphate backbone of the DNA polymer coordinates with the metal cations of the LDH lattice via the ligand-exchange process. This mechanistic insight may facilitate future gene delivery applications using Mg/Fe LDH NPs.



INTRODUCTION Layered double hydroxides (LDHs), a group of anionic inorganic nanocrystals consisting of cationic brucite-like layers and interlayer counteranions, have demonstrated their utility as novel drug- and gene-delivery vehicles as a result of their excellent biocompatibility, high loading capacity, efficient cellular uptake, and pH-based controlled release.1−21 The most studied LDH vehicle for delivery of genes, such as plasmid DNA and oligo-DNA mimics of siRNA, is magnesium aluminum hydroxide (Mg/Al LDH),3,4,17,21−29 which can be described by the formula [Mg(II)1 − xAl(III)x(OH)2]x+(An−)x/n· yH2O, where Mg(II) and Al(III) form the cationic layers and An− serves as an interlayer guest anion and where 0 < x < 1, while y and n are integers. However, mounting evidence indicates that Al can exert neurological, skeletal, and hematological toxicity, making the development of Al-free LDHs capable of maintaining highly efficient gene delivery increasingly desirable. Recently, Mg/Fe LDH nanoparticles (NPs), trademarked as Alpharen and Fermagate, have been vigorously tested in animal and clinical trials for treatment of hyperphosphatemia in hemodialysis patients,30,31 offering solid evidence of their high phosphate removal efficiency and © 2016 American Chemical Society

biocompatibility. We hypothesize that Mg/Fe LDH may bind DNA phosphate backbones in a manner similar to free phosphate, thus being a promising candidate for gene delivery. In this work, we investigate the interface chemistry between DNA and Mg/Fe LDH NPs for the first time. We believe a thorough understanding of the chemistry-regulating interactions at the bio−nano interface will facilitate practical applications of these novel nanomaterials in gene delivery.



RESULTS AND DISCUSSION Surface Properties of Mg/Fe LDH. The as-prepared Mg/ Fe LDHs exhibit a hexagonal nanoplatelet morphology in aqueous solution, with an average size of ∼50 nm, as determined by transmission electron microscopy (TEM) imaging (Figure 1A), and hydrodynamic size of ∼80 nm, as determined by dynamic light scattering (DLS) measurement (Figure 1B). This relatively small LDH NP size confers

Received: December 20, 2015 Revised: February 20, 2016 Published: February 26, 2016 2659

DOI: 10.1021/acs.langmuir.5b04643 Langmuir 2016, 32, 2659−2667

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Langmuir

Figure 1. Physical characterization of Mg/Fe LDH NPs by (A) TEM, (B) DLS, (C) XRD, and (D) ξ potential measurement.

Figure 2. (A and B) Mg/Fe LDH NPs quench the fluorescence of FAM−DNA upon adsorption. The instantaneous adsorption of DNA on LDH surfaces characterized by (C) fluorescence measurement and (D) XRD.

advantages for biomedical applications: smaller sized NPs demonstrate superior colloidal stability and higher cellular uptake than larger LDH NPs.32 The crystal structure of LDH was characterized using X-ray diffraction (XRD; see results in Figure 1C). To understand the interaction of LDH with negatively charged DNA molecules, we studied the surface charge of the LDH NPs by measuring their ξ potential in water and various buffers. The data (Figure 1D) demonstrated that native LDH NPs were highly positively charged as a result of the presence of Fe3+ in the crystal lattice of the nanoplatelets.33,34 Their ξ potential decreased with the increasing solution pH, with the point of zero charge (pzc) at pH 12. However, charge polarity of LDH NPs was reversed to negative in phosphate solutions (pH 5−11) and slightly negative in 4(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.6), Tris−HCl buffer (pH 7.6), and citrate solution (pH 4−11). In acetate buffers (pH 3−5), LDH NPs lost a large portion of the positive charges but maintained a net positive charge. These results showed the susceptibility of LDH

NPs to anions in solution, especially phosphate anions, indicating the high affinity of phosphate ions to the Mg/Fe LDHs. DNA Adsorption. We quantitatively evaluated DNA adsorption by fluorescence measurement using fluorescent dye (e.g., 6-carboxyfluorescein and FAM) labeled DNA, because Mg/Fe LDHs were witnessed to quench fluorescence (panels A and B of Figure 2), similar to gold NPs, carbon nanotubes, graphene, nanoceria, titanium oxide, and iron oxide magnetic NPs, providing a convenient analytical means to investigate DNA adsorption and desorption.35−39 First, the effect of different DNA sequences on the adsorption kinetics was studied. Herein, we compared the adsorption of four different homo-DNA (FAM−A15, FAM−T15, FAM−C15, and FAM−G15) and one random sequence, a 24-mer DNA consisting of four different nucleotides. As shown in Figure 2C, all of the DNA sequences adsorbed to LDH instantaneously. The ultrafast adsorption kinetics suggests that the adsorption essentially occurred on the outside surface of the 2660

DOI: 10.1021/acs.langmuir.5b04643 Langmuir 2016, 32, 2659−2667

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Figure 3. (A) Effect of DNA length on the adsorption capacity of DNA. (B) Adsorbed number of nucleotide footprints on LDH NP surfaces. (C) Adsorption isotherm of FAM−24-mer DNA. (D) DNA length effect on DNA adsorption kinetics.

of polyA, i.e., AMP) were linearly proportional (Figure S2B of the Supporting Information; regression slope = 82.3, and R2 = 0.9949); however, when the total number of nucleotides were in excess relative to binding sites (as with A30 and A45 adsorption), the total number of nucleotides (i.e., AMP units) adsorbed were roughly similar (∼1920 AMPs for A30 and ∼2070 AMPs for A45, as shown in Figure 3B), indicating saturation of surface binding sites. Surface saturation was further evidenced by studying the adsorption isotherm with different concentrations of FAM−24-mer DNA solution (Figure 3C). The perfect Langmuir isotherm fit indicates that the DNA adsorption is reversible and stops once a monolayer is formed on LDH surfaces. However, adsorption kinetics were unaffected by DNA length (Figure 3D), again suggesting that adsorption is constrained to the outer surfaces of LDH NPs, with insignificant adsorption, if any, occurring in the interlayer space. Next, the effect of the ionic strength on DNA adsorption was studied by varying NaCl concentrations in the DNA solutions prior to adding LDH NPs. NaCl did not affect the binding kinetics but significantly increased FAM−G15 adsorption capacity (i.e., from 50% adsorbed to 80%) (Figure S3 of the Supporting Information). Presumably, adsorption capacity increased by shrinking the G15 physical footprint occupied on the NP surface (compact secondary structures resulting in low loading capacity compared to the other homo-DNA) as a result of decreased intramolecular charge repulsion by screening the charge of their phosphate backbones. In contrast, NaCl did not increase the loading capacity of other homo-DNA significantly, which is understandable considering their already high loading capacity in the absence of NaCl. We compared the adsorption of single-stranded DNA (ssDNA) with double-stranded DNA (dsDNA) by adding LDH solutions to either: (1) FAM−A15 (5 nM) with differing amounts of A15 (without the FAM label) in the plate wells or (2) FAM−A15 (5 nM) mixed with various amounts of T15 (complementary sequence to A15, without the FAM label),

NPs without time-consuming diffusion into the interlayer pores. This understanding was supported by the XRD structural analysis of LDH. No interlayer distance increase was observed by comparing the XRD patterns of LDH NPs before and after mixing with various DNA strands and even their structural units, including single-stranded oligo-DNA, single-stranded genomic DNA, double-stranded genomic DNA, nucleoside, and nucleobases, thus indicating that no intercalation occurred (Figure 2D and Figure S1 of the Supporting Information). Such near-instantaneous adsorption, attributable to the charge attraction between the negatively charged DNA and positively charged LDHs, was thermodynamically spontaneous because there was no energy barrier impeding the DNA adsorption. This differs from DNA adsorption to gold or Fe3O4 NPs, where charge repulsion between DNA and the NPs forms an energy barrier, which must be overcome with the assistance of salt or low-pH buffer.40−44 However, adsorption capacity is affected by the sequence composition. For example, significantly less G15 was adsorbed than the other three 15-mer DNA and the 24-mer. The reduced loading capacity of LDH NPs toward G15, similar to that observed for TiO2 and Fe3O4 NPs,45,46 is attributed to secondary structures formed by G15 sequences.47,48 Second, the effect of DNA length on adsorption was investigated. DNA loading capacity decreased for longer DNA sequences, demonstrating that DNA adsorption was limited by the NP surface area (Figure 3A and Figure S2A of the Supporting Information). If we assume adsorbed DNA lies “flat” on the NP surfaces, each DNA nucleotide will create a length-dependent footprint on the surface. This assumption was confirmed by plotting the total number of adsorbed adenosine monophosphate (AMP) units over the DNA length (Figure 3B), where the saturation of the surface area by FAM− polyA strands of the same concentrations but various lengths was demonstrated. For example, when the potential LDH surface binding sites were in excess (as in the case of adsorbing A5 and A15), the numbers of their footprints (the structural unit 2661

DOI: 10.1021/acs.langmuir.5b04643 Langmuir 2016, 32, 2659−2667

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Figure 4. Comparison of the adsorption of (A) dsDNA with (B) ssDNA.

Scheme 1. Analogue Displacement Experiment To Identify the DNA Functionality Contributing to Its Adsorption on Mg/Fe LDH NPs

analogues that share partial structural similarities with DNA were tested for their competitive adsorption onto LDHs. As shown in Scheme 1, analogues included nucleobases, glucose, fructose, and peptide nucleic acid (PNA, specifically pA15 herein). The results (panels A and B of Figure 5) showed that DNA adsorption was not affected by the presence of pre-added nucleobases (final concentration of 1 mM) or sugars (10 mM) in LDH solution, even considering that their concentrations were 200 000 times higher than that of 5 nM DNA, indicating that neither sugars nor bases were responsible for DNA adsorption. Rather, two independent lines of evidence revealed that it is the phosphate backbone of DNA that anchors the molecule onto LDH surfaces. First, we compared the adsorption of a peptide nucleic acid (FAM−pA15) and its DNA counterpart FAM−A15, because pA15 and A15 are very similar in their molecular structure, except their backbone, where pA15 uses an amide backbone, while A15 uses a phosphate backbone, to link the 15 deoxyadenosines. FAM− pA15 did not adsorb to LDH NPs, while the same concentration of FAM−A15 adsorbed near instantaneously upon LDH NP addition to the DNA solution (Figure 5C). The slow decrease

along with 150 mM NaCl into both ssDNA and dsDNA samples to facilitate DNA hybridization in dsDNA samples. The data (Figure 4) showed that LDH NPs adsorbed approximately the same amount of FAM−A15 in the presence or absence of T15, although adsorption kinetics of FAM−A15 slowed when the concentrations of T15 were in far excess (e.g., 100 or 200 times that of FAM−A15) relative to FAM−A15. Competitive inhibition of formed dsDNA (FAM−A15−T15) by excess ssDNA (T15, in Figure 4A) was not as significant as in the system with only ssDNA (FAM−A15 with non-labeled A15 in Figure 4B). Such competition was also apparent in the same systems without NaCl where only ssDNA was present (Figure S4 of the Supporting Information). Identification of DNA Molecular Functionality Entity Responsible for Adsorption by Analogue Displacement. DNA molecules, from a functional copolymer perspective, are composed of nucleotide structural monomers, each of which consists of one nucleobase [cytosine (C), guanine (G), adenine (A), or thymine (T)], a deoxyribose sugar, and a phosphate group linking the nucleotide monomers and forming the backbone of the DNA molecule.49,50 To identify which DNA molecule functional entity contributes to adsorption, several 2662

DOI: 10.1021/acs.langmuir.5b04643 Langmuir 2016, 32, 2659−2667

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Figure 5. Effect of (A) pre-existed nucleobases, (B) glucose/fructose, (C) phosphate backbones (from PNA), and (D) various anions on the adsorption of DNA on LDH NP surfaces.

in the PNA fluorescence signal was due to its non-specific adsorption to the plate well surface because PNA is more hydrophobic than DNA molecules.51,52 Nevertheless, this result substantiated that DNA adsorption on LDH surfaces is based on its phosphate backbone. The second line of evidence came from the studies on the effect of phosphate anions on DNA adsorption and desorption. When FAM−A15 is added to the LDH aqueous solution (without other anions added), only 90% DNA adsorbed by LDHs. However, when 2 mM phosphate anions were pre-added in the LDH solution, ∼85% fluorescence remained, indicating the presence of phosphateinhibited adsorption of FAM−A15 to a significant extent. A more detailed study revealed that such phosphate inhibition is concentration-dependent (Figure S5 of the Supporting Information). The potential contribution of the Na+ cations in the system was excluded by investigating the effect of NaCl on DNA adsorption (Figure S3 of the Supporting Information). When we tested the effect of other anions (CH3COO−, Cl−, S2O8−, HCO3−, AsO33−, and AsO43−), only AsO43−, an analogue of phosphate ions, showed similar but less inhibition (∼20% fluorescence remained; Figure 5d) on DNA adsorption. These results suggested that phosphate anions share the same binding sites on LDH surfaces with DNA, thus resulting in inhibition of DNA adsorption by competition for the binding sites. Further, we tested the phosphate-induced DNA desorption from LDH surfaces. As shown in Figure S6 of the Supporting Information, the pre-adsorbed FAM−DNA can be desorbed from LDH surfaces in phosphate buffer. A higher concentration of phosphate induced faster DNA desorption, evidenced by the more rapid increase of the fluorescence intensity over time. In addition, the desorption kinetics of DNA with different sequences were different, in the order of G15 < A15 < C15 < T15, which may suggest some interactions of the nucleobases with LDH NPs (vide inf ra), even though the base−LDH

interaction is weak compared to phosphate backbone adsorption. In a quantitative analysis of the free phosphateinduced DNA desorption, we observed that, only when the concentrations of added phosphate anions are 105 times higher than the total concentration of the phosphate group in the total adsorbed DNA strands (i.e., ∼20 mM for free phosphate versus 75 nM DNA phosphate backbone), the replacement was significant (50% adsorbed DNA replaced), indicating the formation of strong interactions between phosphate backbones of DNA and LDH surfaces. Such a strong interaction suggests that DNA adsorption on Mg/Fe LDHs is a chemisorption rather than physical adsorption, mainly as a result of metal coordination between the phosphate backbone and the metal ions in LDH NPs.53 In addition, as a polymer with multiple phosphate groups, the multivalent binding of DNA strands may contribute to such strong binding to LDHs.54,55 Identification of the DNA Binding Sites on LDH Surfaces. To identify the active binding sites of Mg/Fe LDH that adsorb DNA strands, we studied DNA adsorption on MgO and Fe2O3 NPs in parallel, which might provide insight into dissecting the unique contribution of each of the metal ions in Mg/Fe LDH to DNA adsorption. Similar to previous studies,46 we observed FAM−DNA adsorbed on Fe2O3 NPs, resulting in fluorescence quenching in the presence of 300 mM NaCl (data not shown because similar data were published previously). We also noticed significant FAM−DNA adsorbed on MgO NPs, even without NaCl added, attributable to the positively charged MgO NPs (ξ potential = 23.2 ± 1.6 mV in pure water), attracting DNA by electrostatic attraction. We further tested the effect of various anions and nucleobases on adsorption and desorption of FAM−DNA on MgO NP surfaces. The results showed that DNA−MgO binding is even stronger than the DNA−LDH interaction. Among various pre-added anions and nucleobases (10 mM) in MgO solutions (1.8 mg/mL), only carbonate, phosphate, and citrate anions showed a slight influence on DNA adsorption (