Self-Assembly of DNA−Minocycline Complexes by Metal Ions withControlled Drug ReleaseTing Zhang,†,‡,# Jia Nong,§,# Nouf Alzahrani,‡,# Zhicheng Wang,§ Sung Won Oh,† Tristan Meier,∥

Dong Gyu Yang,‡ Yonggang Ke,⊥ Yinghui Zhong,*,§ and Jinglin Fu*,†,‡

†Center for Computational and Integrative Biology, Rutgers UniversityCamden, 315 Penn Street, Camden, New Jersey 08102,United States‡Department of Chemistry, Rutgers UniversityCamden, 315 Penn Street, Camden, New Jersey 08102, United States§School of Biomedical Engineering, Science and Health Systems, Drexel University, 3141 Chestnut Street, Philadelphia,Pennsylvania 19104, United States∥Eastern Regional High School, 1401 Laurel Oak Road, Voorhees, New Jersey 08043, United States⊥Wallace H. Coulter Department of Biomedical Engineering, Emory School of Medicine, 1760 Haygood Drive, Atlanta, Georgia30322, United States

*S Supporting Information

ABSTRACT: Here we reported a study of metal ions-assistedassembly of DNA−minocycline (MC) complexes and theirpotential application for controlling MC release. In thepresence of divalent cations of magnesium or calcium ions(M2+), MC, a zwitterionic tetracycline analogue, was found tobind to phosphate groups of nucleic acids via an electrostaticbridge of phosphate (DNA)-M2+-MC. We investigatedmultiple parameters for affecting the formation of DNA-Mg2+-MC complex, including metal ion concentrations, basecomposition, DNA length, and single- versus double-strandedDNA. For different nitrogen bases, single-stranded poly(A)20and poly(T)20 showed a higher MC entrapment efficiency ofDNA-Mg2+-MC complex than poly(C)20 and poly(G)20. Single-stranded DNA was also found to form a more stable DNA-Mg2+-MC complex than double-stranded DNA. Between different divalent metal ions, we observed that the formation of DNA-Ca2+-MC complex was more stable and efficient than the formation of DNA-Mg2+-MC complex. Toward drug release, we usedagarose gel to encapsulate DNA-Mg2+-MC complexes and monitored MC release. Some DNA-Mg2+-MC complexes couldprolong MC release from agarose gel to more than 10 days as compared with the quick release of free MC from agarose gel inless than 1 day. The released MC from DNA-Mg2+-MC complexes retained the anti-inflammatory bioactivity to inhibit nitricoxide production from pro-inflammatory macrophages. The reported study of metal ion-assisted DNA-MC assembly not onlyincreased our understanding of biochemical interactions between tetracycline molecules and nucleic acids but also contributedto the development of a highly tunable drug delivery system to mediate MC release for clinical applications.

KEYWORDS: DNA nanostructure, minocycline, DNA-minocycline assembly, controlled drug release, anti-inflammation

1. INTRODUCTIONDNA is a “Popstar” molecule in biochemistry that carriesgenetic information to guide the construction of biomolecularcomplexes in living systems. In the past three decades, self-assembled DNA nanostructures have emerged as promisingbiomaterials to organize molecules on the nanoscale.1−3 Theuse of double-helical DNA molecules for nanoscale engineer-ing began with Seeman’s construction of an artificial branchedDNA “Holliday” junction.4 Recent breakthroughs in scaffoldedDNA origami5 and single-stranded DNA bricks6 haveempowered the design and fabrication of a virtually unlimitedreservoir of spatially addressable one-dimensional (1D), 2D,and 3D nanostructures,7,8 including structures with complexcurvatures,9,10 polyhedral meshes,11,12 and periodic DNA

crystals.13,14 DNA scaffold-directed assembly holds greatpromise to organize bioactive therapeutics with precise controlof spatial patterning.57−60 Self-assembled DNA nanostructurescan serve as delivery platforms that are integrated with variousfunctions ranging from molecular recognition and computa-tions; dynamically structural switch to carry molecularpayloads; and selectively release.15−20 Single- or double-stranded DNA (ssDNA or dsDNA) can also bind to smalldrug molecules and deliver them into cells for therapy, such asdoxorubicin, daunorubicin, and epirubicin.21

Received: May 9, 2019Accepted: June 5, 2019Published: August 9, 2019

Research Article

www.acsami.orgCite This: ACS Appl. Mater. Interfaces 2019, 11, 29512−29521

© 2019 American Chemical Society 29512 DOI: 10.1021/acsami.9b08126ACS Appl. Mater. Interfaces 2019, 11, 29512−29521

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Tetracycline molecules are broad-spectrum antibiotic drugsthat are used to clinically treat various bacterial infections andare also applied to the treatment of rheumatoid arthritis and inthe prevention of malaria.22 The structural analysis hasrevealed that tetracycline molecule can bind to ribosomal30S subunit stabilized by magnesium salt bridge betweenhydroxyl groups of tetracycline and phosphate groups ofrRNA, and thus inhibited ribosome activity.22,23 This structuralinformation suggests a possibility that tetracycline moleculesmay interact with nucleic acids and could be assembled totetracycline/nucletotide hybrid nanostructures. Tetracylinewas also reported to bind with Calf thymus DNA in thepresence of Cu2+.24 Minocycline (MC), a semisynthetictetracycline analogue, is a clinically available antibiotic andanti-inflammatory drug that has a broader antibiotic spectrumthan other members of the tetracycline group.25−27 In addition,MC also demonstrates potent antioxidant and antiapoptoticproperties.28−30 Due to its multifaceted activities, MC has beenshown to target a number of debilitating neurological diseases,including traumatic spinal cord injury,28,31 brain injury,32

stroke,33 intracerebral hemorrhage,34 Parkinson’s disease,35

Alzheimer’s disease,36 multiple sclerosis,37 and amyotrophiclateral sclerosis,38 etc. However, the effectiveness of MCtreatment for these disorders has been compromised by theinability to locally deliver high concentrations of this drug.39

To mediate its local delivery in tissue, people have developedvarious MC-hybrid particles for encapsulation and release ofMC, such as hydrophobic poly(lactic-co-glycolic acid) (PLGA)microspheres40,41 and dextran sulfate-MC nanoparticles.39,42,43

Here, we reported a study of metal ions (M2+)-assisted self-assembly of DNA-M2+-MC hybrid structures. In the presenceof divalent cations of magnesium and calcium, MC self-assembled with various DNA nanostructures to formcoaggregated particles. The DNA-Mg2+-MC particles werefurther used to control MC release from agarose hydrogel andto maintain its anti-inflammatory effect.

2. MATERIALS AND METHODS2.1. Materials. Analytical grade or molecular biology reagents and

distilled (DI) water were used in all experiments unless otherwisespecified. Magnesium acetate (MgAc, Mg(CH3COO)2·4H2O),calcium acetate (CaAc, Ca(CH3COO)2·H2O), Tris base, EDTA,DNA grade water, and agarose were obtained from Fisher Scientific(Pittsburgh, PA, USA). All oligonucleotides were purchased fromIntegrated DNA Technologies (Coralville, IA, USA), and theirsequences were listed in the Supporting Information Table S1. DNAoligonucleotides were dissolved in DNA grade water, and theconcentrations of DNA stock solutions were quantified by 260 nmabsorbance using NanoDrop 2000 (Thermo Scientific, Waltham, MA,USA). Minocycline hydrochloride, adenosine, Griess reagent, lip-opolysaccharide, and parafilm were purchased from Sigma-Aldrich(St. Louis, MO, USA). A 10 mg/mL amount of MC solution wasprepared by dissolving MC powder in DI water prior to use.M13mp18 circular ssDNA was purchased from Affymetrix (Cleve-land, OH, USA). Acetic acid was purchased from Acros Organics(Geel, Belgium). Fetal bovine serum, penicillin, and streptomycinwere ordered from Life Technologies (Carlsbad, CA, USA).Dulbecco’s modified Eagle’s medium was purchased from Corning(Corning, NY, USA). RAW264.7 murine macrophages were kindlyprovided by Dr. Narayan Avadhani from the Department of AnimalBiology at University of Pennsylvania.2.2. Preparation of Buffer Solutions. Buffer solutions were

prepared using DI water and were stocked at 4 °C in dark. A 50 × TAstock solution contains 2 M Tris base and 1 M acetic acid. The pHvalue was adjusted to 7.0 using acetic acid. A 10 × TA solution was

diluted from 50 × TA with the final concentration of 0.4 M Tris. A 1× TA buffer was diluted from 10 × TA with the final concentration of0.04 M Tris. A 1 × TA buffer with 32 mM Mg2+ was prepared byadding 5 mL of 10 × TA stock and 16 mL of 100 mM MgAc into DIwater for the total volume of 50 mL. Solutions of 1 × TA-Mg2+

containing 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 mM Mg2+ were preparedby the serial dilution (2-fold) of 1 × TA with 32 mM Mg2+ using the 1× TA solution. Buffer of 1 × TAE-Mg2+ contains 40 mM Tris, 20 mMacetic acid, 2 mM EDTA, and 12.5 mM MgAc, and the pH value wasadjusted to 8.0. TA buffer solutions with Ca2+ were prepared similarlyusing CaAc instead of MgAc as described above.

The 2 × Hank’s balanced salt solution (HBSS) was prepared aspreviously described that contains 0.8 g/L KCl, 0.12 g/L KH2PO4, 0.4g/L MgSO4·7H2O, 16 g/L NaCl, 0.096 g/L Na2HPO4, 0.7 g/LNaHCO3, and 0.28 g/L CaCl2.

42 The pH value was adjusted to 7.0.The 1 × HBSS solution was prepared by adding 50 mL of 2 × HBSSinto 50 mL of DI water for a total volume of 100 mL.

2.3. Preparation of DNA Sample. ssDNA samples were directlyprepared from stock solutions by diluting them into 1 × TA-Mg2+

buffer. dsDNA samples were preincubated in 1 × TA-Mg2+ buffer atroom temperature for a half-hour prior to the addition of MC. Two-strand multidomain DNA hydrogel was prepared as previouslyreported.44 Briefly, 10 mg/mL solutions of Hydro-1 and Hydro-2were prepared in 1 × TA with 10 mM MgAc separately. The twosolutions were then mixed together at a 1:1 volume ratio that wasthermally annealed to form a 10 mg/mL two-strand multidomainDNA structure. DNA origami rectangular tiles were prepared aspreviously described.45,46 Briefly, 20 nM M13mp18 DNA wasincubated with a 5-fold molar excess of staple strands in 1 × TAE-Mg2+ buffer. The mixture was heated to 95 °C and then cooled to 4°C with a temperature gradient (Supporting Information Table S2).The origami tiles were purified using Amicon filters (MWCO, 100kDa; 500 μL) to remove excess staple strands. The concentration ofDNA origami tiles was quantified by measuring the absorbance at 260nm, assuming an extinction coefficient of ∼109,119,009 M−1 cm−1.

2.4. Preparation of DNA-Mg2+/Ca2+-MC Complexes. A 4 mg/mL DNA solution was first mixed with a 4 mg/mL MC solution in 1× TA buffer with 1:1 volume ratio to produce a 2 mg/mL mixturesolution of DNA and MC. Then, an equal volume of 2 × Mg2+ or 2 ×Ca2+ solution prepared in 1 × TA buffer was added into the mixturesolution of DNA and MC to produce a 1 mg/mL DNA-MC mixtureat desired Mg2+ concentration. The mixture was incubated on theorbital shaker (50 rpm; ArmaLab, Bethesda, MD, USA) at roomtemperature for 30 min in the dark.

2.5. Quantification of the Entrapment Efficiency of DNAand MC in DNA-Mg2+/Ca2+-MC Complexes. A 50 μL aliquot ofDNA-Mg2+/Ca2+-MC complexes was prepared as described in section2.4. The solution was then centrifuged at 10,000 rpm under roomtemperature for 5 min. DNA-Mg2+/Ca2+-MC complexes wereprecipitated on the bottom of the centrifuge tube. A 32 μL aliquotof supernatant solution was collected and diluted by 10-fold into 1 ×TA buffer. The absorbance spectrum of the diluted supernatant wasmeasured using a 96-well Cytation 3 plate reader (Biotek, Winooski,VT, USA). The concentrations of DNA and MC were characterizedby the absorbance at 260 and 355 nm, respectively. The MCabsorbance at 355 nm was affected little by magnesium concen-trations and was used to characterize MC concentration (SupportingInformation Figures S1 and S2). The detailed calculation of MC andDNA entrapment efficiency within DNA-Mg2+/Ca2+-MC complexeswas described in the Supporting Information. The accuracy of theabsorbance analysis of MC supernatant was also verified by theparticle dissolution of DNA-Mg2+-MC complexes (SupportingInformation Figure S3).

2.6. Atomic Force Microscope Imaging. A 2 μL aliquot ofsupernatant solution was deposited onto a freshly cleaved micasurface (Ted Pella, Redding, CA, USA) and was left to adsorb for 2min. Then, 80 μL of 1 × TA buffer was added to the sample and 2 μLof 100 mM Ni2+ was added to enhance DNA adsorption on mica.Then, the buffer solution was removed, and the surface was rinsedusing DI water for three times. After the mica surface was dried under

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clean air, the samples were scanned with SCANASYST-Air probe(Bruker, Billerica, MA, USA) using “Scanasyst in air mode” ofMultimode 8 atomic force microscope (AFM; Bruker).2.7. MC Release from DNA-Mg2+/Ca2+-MC Complexes

Encapsulated in Agarose Gel. DNA-Mg2+/Ca2+-MC complexeswere encapsulated in 1.5% agarose gel for releasing MC in 1 × HBSSbuffer. Please see Supporting Information for detailed procedures.The released MC in milligrams per milliliter was calculated from astandard curve of MC concentrations (Supporting Information FigureS2).The kinetical fitting of MC release curves was performed using

GraphPad Prism 7. A simplified Weibull model was used to fit theexponential profile of cumulative release curves as shown in eq 1:47,48

Q /% (1 e ) 100kTcumulative = − ×−

(1)

where Qcumulative is the cumulative drug release percentage, k is therelease constant, and T is the release period.2.8. MC Bioactivity Test. Anti-inflammatory bioactivity of MC

was tested on RAW264.7 murine macrophages. The cells werecultured in Dulbecco’s modified Eagle’s medium, supplemented with

10% fetal bovine serum and 1% penicillin/streptomycin. Lip-opolysaccharide (LPS) at 250 pg/mL was used to stimulatemacrophages for the upregulation of nitric oxide (NO), a pro-inflammatory mediator.49 Fresh MC (0.5 μg/mL) or released MC(diluted to 0.5 μg/mL) was added to the LPS-stimulated cultures.After 48 h, NO production was determined by the measurement ofaccumulated nitrite (an indicator of NO) in the culture medium usingGriess reagent,42 which produced an increased absorbance at ∼540nm (Supporting Information Figure S4). All data were presented asmean ± standard deviation (S.D.). Pairwise comparisons wereconducted by using one-way ANOVA and Tukey test. p < 0.05 wasconsidered statistically significant.

3. RESULTS AND DISCUSSIONMC has four rings that are similar to tetracycline (Figure 1A)with multiple pKa values for different functional groups: pKa ∼2.8 for the hydroxyl group at C3, pKa ∼ 5 for thedimethylamine group at C7, pKa ∼ 7.8 for the hydroxylgroup at C12, and pKa ∼ 9.5 for the dimethylamine group atC4.50 MC is zwitterionic within the pH range of 5−7.8, with

Figure 1. Metal ions-assisted self-assembly of DNA-Mg2+-MC complexes. (A) Chemical structure of MC. (B) Proposed structural formation ofDNA-Mg2+-MC complex. Mg2+ serves as a coordination bridge between the hydroxyl group of MC and the phosphate group of nucleotides; a weakπ−π interaction between DNA bases and the aromatic ring of MC may also exist. (C) Macroscopic observation of Mg2+-assisted self-assembly andprecipitation of DNA-MC complexes. Condition: 1 mg/mL poly(A)20 and 1 mg/mL MC in 1 × TA (pH 7) buffer with 0, 0.25, 0.5, 1, 2, 4, 8, and16 mM Mg2+. (D) Absorption spectra of supernatants of DNA-Mg2+-MC solutions. Both DNA and MC absorption peaks decrease as the additionof more Mg2+. (E) AFM imaging of DNA-Mg2+-MC complexes for small nanoparticles (top) and large aggregates (bottom). The aggregates wereprepared by mixing 1 mg/mL poly(A)20 with 1 mg/mL MC in pH 7, 1 × TA with 4 mM Mg2+. Scales: 1 μm (top) and 5 μm (bottom).

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one negative charge from the deprotonation of the hydroxylgroup at C3 and one positive charge from the protonateddimethyl amine at C4.50 Based on the previous studies oftetracycline interacting with rRNA,22,23 it is suggested thatMg2+ might form a salt bridge between the hydroxyl group onMC and the phosphate group on nucleotides, serving as ametal bridge to assemble MC and nucleic acids together(Figure 1B). A weak π−π interaction between DNA bases andthe aromatic ring of MC may also exist to stabilize DNA-MCassemblies. To test this hypothesis, we first titrated Mg2+

concentrations to affect the assembly of a ssDNA with MC insolution. As shown in Figure 1C, the mixture of poly(A)20 andMC is a yellow solution without any observed precipitant at 0mM Mg2+. As gradually increasing Mg2+ concentration from 0to 16 mM, the solution started to become turbid and yellowprecipitants were observed at higher Mg2+ concentrations. Thespectrum analysis indicated that there was a decreasedabsorbance for both MC (355 nm) and DNA (260 nm) insolution supernatant with the addition of Mg2+ (Figure 1D),suggesting less MC and DNA were left in the solution layer. Itindicated that MC and DNA were coaggregated intoprecipitants assisted by magnesium ion. Mechanistic informa-tion given by fluorescence spectrum and infrared (IR) analysis

supported our hypothesis of a Mg2+ bridged DNA-Mg2+-MCassembly (Supporting Information Figures S5 and S6). Asshown in Figure 1E, AFM imaging of DNA-Mg2+-MCcomplexes showed that tiny nanoparticles (∼100 nm, topimage) were first formed in the presence of magnesium. Thesetiny nanoparticles further aggregated into large particles of afew micrometers size (bottom image). The formation of DNA-Mg2+-MC was also characterized by dynamic light scattering(Supporting Information Figure S7).To evaluate the capability of DNA for interacting with MC,

we first evaluated the effect of ssDNA length on the formationof DNA-Mg2+-MC complexes. As shown in Figure 2A, MCentrapment efficiency in DNA-Mg2+-MC complexes reachedmaximum at ∼4 mM Mg2+ for all poly(A) strands with lengthvarying from 11 nucleotides (nt) to 50 nt, suggesting thesaturation of Mg2+ in DNA-Mg2+-MC complexes. As a control,the single nucleotide, adenosine, formed little particles withMC with a very low MC entrapment efficiency. Length analysisshowed that MC entrapment efficiency increased as the DNAlength increased from 11 nt (55%) and 15 nt (62%) to 20 nt(74%). For poly(A) longer than 20 nt, the MC entrapmentefficiency did not increase significantly (Figure 2B). As shownin Figure 2C, the molar ratio of MC to monomer nucleotide in

Figure 2. Formation of DNA-Mg2+-MC complexes depending on the length of ssDNA. (A) Titration of Mg2+ concentrations for MC entrapmentefficiency of different lengths of poly(A) ranging from 11 to 50 nt and adenosine. (B) MC entrapment efficiency of different lengths of poly(A)ranging from 11 to 50 nt at 4 mM Mg2+. (C) Ratios of MC to nucleotide monomer in DNA-Mg2+-MC complexes. All experiments were performedfor 1 mg/mL DNA and 1 mg/mL MC in pH 7, 1 × TA buffer. Error bars: the range of data for three replicates.

Figure 3. Formation of DNA-Mg2+-MC complexes for poly(A)20, poly(T)20, poly(C)20, and poly(G)20 with the evaluation of (A) MC entrapmentefficiency, (B) DNA entrapment efficiency, and (C) ratios of MC to nucleotide monomer. All of the experiments were performed for 1 mg/mLDNA and 1 mg/mL MC in pH 7, 1 × TA buffer with 4 mM Mg2+. Error bars: the range of data for three replicates. *, p < 0.05 compared withpoly(A)20 or poly(T)20 (n = 3).

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DNA-MC particles was ∼0.8−0.9 for these poly(A) strands,except for poly(A)15 with a ratio >1. This indicated that onenucleotide could bind with almost one MC on average inssDNA-Mg2+-MC complexes.We compared different bases of A, T, C, and G for their

ability to form DNA-Mg2+-MC complexes. As shown in Figure3A, poly(A)20 (∼74%) and poly(T)20 (∼70%) entrapped moreMC in DNA-MC particles than poly(C)20 (∼62%) andpoly(G)20 (∼64%). However, in DNA-Mg2+-MC complexes,more DNA were entrapped in the solid precipitants forpoly(C)20 (>90%) and poly(G)20 (∼100%) than poly(A)20(∼45%) and poly(T)20 (∼45%) (Figure 3B). As a result, themolar ratio of MC to monomer nucleotide in DNA-MCcomplex was ∼0.4 for poly(C)20 and poly(G)20, only half ofthe ratio (∼0.8−0.9) for poly(A)20 and poly(T)20 (Figure 3C).One possible reason is that poly(C)20 and poly(G)20 presentpartially folded structures in solution (e.g., cytosine-cytosine+

pair in i-motif structures51 and G4 interactions in G-quadruplex52) and thus bind less MC than relatively linearpoly(A)20 and poly(T)20. In addition, we performed isothermaltitration of DNA concentrations for entrapping MC (Support-ing Information Figure S8) and pH titrations from 4 to 8(Supporting Information Figure S9).We also tested dsDNA for forming the DNA-Mg2+-MC

complex. As shown in Figure 4A, the MC entrapmentefficiency reached maximal at 4 mM Mg2+ (∼60%) for thepoly(A-T)20-Mg2+-MC complex, and then quickly decreasedwhen Mg2+ concentrations further increased. In contrast,poly(A)20 entrapped more MC (>70%) than poly(A-T)20 inthe assembled complex at the same Mg2+ concentration, andthe MC entrapment efficiency did not show significantdecrease at higher Mg2+ concentrations of 8 and 16 mM.Figure 4B showed that the DNA entrapment efficiency ofpoly(A-T)20 was also lower than that of poly(A)20 in DNA-

Figure 4. Formation of DNA-Mg2+-MC complexes for ssDNA and dsDNA with the evaluation of (A) MC entrapment efficiency depending onMg2+ concentrations, (B) DNA entrapment efficiency depending on Mg2+ concentrations, and (C) ratios of MC to nucleotide monomer for DNA-Mg2+-MC complexes at 4 mM Mg2+. All of the experiments were performed for 1 mg/mL DNA and 1 mg/mL MC in pH 7, 1 × TA buffer. Errorbars: the range of data for three replicates.

Figure 5. MC entrapment efficiency for DNA-Mg2+-MC complexes formed by different DNA nanostructures, including (1) poly(A)20 and poly(A-T)20, (2) poly(A)40 and poly(A-T)40, (3) multidomain ssDNA and two-strand multidomain DNA hydrogel, and (4) M13mp18 ssDNA andrectangular DNA origami. All of the experiments were performed for 1 mg/mL DNA and 1 mg/mL MC in pH 7, 1 × TA buffer with 4 mM Mg2+.Error bars: the range of data for three replicates.

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Mg2+-MC complexes. A similar pattern of decreased DNAentrapment was also observed for poly(A-T)20 at higher Mg2+

concentrations of 8 and 16 mM. However, the overall molarratio of MC-to-monomer nucleotide in assembled complexeswas similar for all ssDNA and dsDNA strands (Figure 4C).dsDNA is more rigid in structure than flexible ssDNA, where

divalent magnesium ion binds tighter with dsDNA thanssDNA. The Mg2+-dsDNA interaction can shield the negativecharge of backbone phosphate and stabilize double-helixstructures. However, this also reduces the electrostaticinteraction of the MC-Mg2+-phosphate (DNA) bridge. Thisreduction is significant at high magnesium concentrations (e.g.,

Figure 6. Formation of DNA-M2+-MC complexes for Mg2+ and Ca2+ with the evaluation of (A) MC entrapment efficiency of poly(A)20-MCcomplexes depending on Mg2+ or Ca2+ concentrations and (B) MC entrapment efficiency of poly(A-T)20-MC complexes depending on Mg2+ orCa2+ concentrations. (C) Reduction of MC entrapment efficiency of poly(A)20-Mg2+-MC complexes due to the addition of Na+. Condition: NaClcompetition was performed for 1 mg/mL poly(A)20 and 1 mg/mL MC in pH 7, 1 × TA buffer with 4 mM Mg2+. Error bars: the range of data forthree replicates.

Figure 7. Cumulative MC releases from various DNA-Mg2+-MC complexes for (A) poly(A)20 encapsulated in agarose gel, poly(T)20, poly(C)20,and poly(G)20, as well as free MC; (B) poly(A)20, poly(A)40, and M13mp18 DNA; (C) ssDNA of poly(A)20 and poly(A)40 and dsDNA of poly(A-T)20 and poly(A-T)40; (D) DNA-Mg2+-MC complex and DNA-Ca2+-MC complex. All DNA-M2+-MC complexes were performed with 1 mg/mLDNA and 1 mg/mL MC in pH 7, 1 × TA buffer with 4 mM Mg2+ or Ca2+. DNA-M2+-MC complexes were entrapped within 1.5% agarose gel andwere released in 1 × HBSS buffer. Error bars: the range of data for three replicates.

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8 mM or higher) at which dsDNA is saturated with Mg2+ ionsand thus is unable to interact with MC efficiently. As shown inFigure 5, we further compared various DNA nanostructures forentrapping MC in DNA-Mg2+-MC complexes. ssDNA ofpoly(A)20 or 40 entrapped more MC (>70%) than rigid dsDNAof poly(A-T)20 or 40 (∼60%). Multibranched dsDNA structures,such as DNA hydrogel assembled from two multidomainssDNA strands,44 improved MC entrapment efficiency to 70%or more. Long and circular M13mp18 ssDNA (7249-nt)entrapped >80% of MC, which was higher than a rectangularDNA origami assembled (∼70%) from double-strandedhybridizations of M13mp18 DNA and staple strands.Besides Mg2+, other divalent metal ions may also assist the

formation of DNA-M2+-MC complexes stabilized by metal ionbridges. As shown in Figure 6A, poly(A)20-Ca

2+-MC complexentrapped 95% of MC in the assembled complex when Ca2+

was 4 mM or higher, whereas the MC entrapment efficiencyfor poly(A)20-Mg2+-MC complex was significantly lower(∼74%) at the same Mg2+ concentrations. For dsDNA ofpoly(A-T)20, a stable poly(A-T)20-Ca

2+-MC complex wasformed with the entrapment of 90% MC when Ca2+ was 4mM or higher, while a poly(A-T)20-Mg2+-MC complex reacheda maximal entrapment of 60% MC at 4 mM Mg2+ and the MCentrapment decreased at higher Mg2+ concentrations > 4 mM(Figure 6B). This result suggested that the overall Ca2+-mediated DNA and MC interaction was stronger than theMg2+-mediated interaction, which led to more MC entrapmentin DNA-Ca2+-MC complexes, especially for a dsDNAmolecule. Ca2+ is larger than Mg2+ in size, which enablesstronger M2+-phosphate interaction in DNA-M2+-MC com-plexes based on Fajans’ rules. However, high concentrations ofcalcium ions are potentially toxic in neural applications. Forexample, Ca2+ can damage cell structures including cytoske-leton, membrane, and chromosome, and induces apopto-sis.39,53 Conversely, Mg2+ has been proved to be neuro-protective from many clinical trials of neural injuries.31,54 Thus,in the following studies, we focused on the use of DNA-Mg2+-MC complex to mediate MC release from agarose hydrogel.

We also found that monovalent cations such as Na+

destabilized the DNA-Mg2+-MC complex. As shown in Figure6C, with the addition of more Na+ into the solution, less MCwas entrapped within poly(A)20-Mg2+-MC complexes due tothe disruption of the Mg2+ bridge under the competition ofNa+. On the other hand, at 160 mM NaCl (higher thanphysiological NaCl concentration of 154 mM), poly(A)20-Mg2+-MC still entrapped more than 50% of MC in precipitantparticles. In addition, the body fluid contains Ca2+ and Mg2+

ions that can stabilize MC release. Therefore, we used HBSSbuffer as the release medium to simulate body fluid.55

Toward local MC delivery, we encapsulated DNA-Mg2+-MCcomplexes in 1.5% (m/v) agarose gel that could be applied toimmobilizing the complexes at the injury/disease site.39 Asshown in Figure 7A, when free MC was loaded in agarosehydrogel, more than 90% of loaded MC was released at day 1,due to the fast diffusion of MC through the macroporoushydrogel. In contrast, agarose hydrogel loaded with ssDNA-Mg2+-MC complex released MC substantially slower: ∼40%and 60% MC were released at day 1 and day 2, respectively,followed by a slow MC release from day 3 to day 10. Toanalyze the release kinetics, a simplified Weibull model wasused to fit the exponential profile of cumulative releasecurves.47,48 Poly(A)20-Mg2+-MC and poly(T)20-Mg2+-MC hadsimilar release constants (k value) of ∼0.43 ± 0.02 and ∼0.44± 0.02, respectively, which were smaller than the releaseconstants of poly(C)20-Mg2+-MC (k ∼ 0.56 ± 0.01) andpoly(G)20-Mg2+-MC (k ∼ 0.50 ± 0.01). This result wasconsistent with our observation of weaker poly(C)20/poly-(G)20-Mg2+-MC interactions as shown in Figure 3. Next, weevaluated how the length of ssDNA affected the MC releasefrom the complexes. As shown in Figure 7B, the release of MCfrom poly(A)40-Mg2+-MC (longer ssDNA) was significantlyslower (k ∼ 0.20 ± 0.01) than that from poly(A)20-Mg2+-MC(shorter ssDNA) (k ∼ 0.43 ± 0.02). The release of MC from7249 nt M13mp18-Mg2+-MC complex was even slower (k ∼0.16 ± 0.01). Figure 7C showed that the release of MC fromdsDNA-Mg2+-MC complexes was faster than that from

Figure 8. Anti-inflammatory bioactivity of (A) released MC and (B) DNA-Mg2+-MC incorporated agarose gel. RAW 264.7 murine macrophageswere incubated with various solutions, including LPS alone, LPS with blank gel, LPS supplemented with fresh or released MC, and LPS with DNA-Mg2+-MC incorporated gel. Cells without any treatment were used as control. Cell culture medium containing released NO was measured in theform of nitrite after 48 h incubation. *, p < 0.05 compared with LPS alone-treated culture (n = 3). Error bar: the range of data for three replicates.

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ssDNA-Mg2+-MC complexes, possibly due to the unstabledsDNA-Mg2+-MC interactions as discussed in Figure 4. Fordifferent divalent ions, the MC release from poly(A)20-Ca

2+-MC (k ∼ 0.14 ± 0.01) was significantly slower than that frompoly(A)20-Mg2+-MC (k ∼ 0.43 ± 0.02). This could beattributed to the enhanced electrostatic interactions of thephosphate (DNA)-Ca2+-MC bridge (Figure 7D). Conversely,the dsDNA poly(A-T)20-Ca

2+-MC complex was unstable,resulting in substantially and significantly faster MC release(k ∼ 0.70 ± 0.01) than that from the ssDNA complex ofpoly(A)20-Ca

2+-MC.To evaluate the anti-inflammatory activity of released MC,

RAW 264.7 murine macrophages were treated with LPS toinduce the pro-inflammatory phenotype of macrophages,which were marked by the upregulation of NO production.56

It has been reported that MC, as an anti-inflammatory drug, isable to inhibit the activation of pro-inflammatory macrophagesand reduce NO production.57 As shown in Figure 8A,significantly more accumulated nitrite (an NO indicator) wasdetected in LPS-stimulated macrophages (nitrite, ∼7.2 ± 0.3μM) compared with untreated control (nitrite, <1 μM). Theaddition of fresh MC significantly reduced the level ofaccumulated nitrite in LPS-induced macrophages to ∼3.4 ±0.2 μM, confirming its anti-inflammatory effect. Fresh MC andMC released from poly(A)20-Mg2+-MC at day 4 or poly(A)40-Mg2+-MC at day 7 showed similar anti-inflammatorybioactivity for suppressing NO production. As shown inFigure 8B, the DNA-Mg2+-MC incorporated agarose gel wasdemonstrated to suppress NO production of macrophages.DNA-Mg2+-MC gels with different release periods (from 3days to 7 days) were also tested for anti-inflammation againstLPS-induced macrophages (Supporting Information FigureS10). These results demonstrated that the released MC fromDNA-Mg2+-MC complexes retained bioactivity similar to thatof fresh MC. To be noted, M13mp18 viral ssDNA and itsscaffolded origami structures were found to strongly induce theinflammatory response of macrophages and were not used tomediate the release of MC (Supporting Information FigureS11). M13mp18 DNA or M13mp18-scaffolded DNA nano-structures have been previously reported for activating a potentinflammatory cytokine response that was similar to theresponse from the exposure to bacterial or viral nucleicacids.58−60

4. CONCLUSIONWe have studied the self-assembly and coaggregation of DNAmolecules with MC stabilized by the divalent metal ion bridgeof Mg2+ or Ca2+. By comparing the interactions of differentDNA nanostructures with MC, we concluded that ssDNA wasmore efficient (e.g., poly(A)20 or poly(A)40) than dsDNA forentrapping MC within DNA-M2+-MC complexes, which wasattributed to the more flexible structures of ssDNA thandsDNA. We also observed that ssDNA of poly(A)20 andpoly(T)20 entrapped more MC with a higher ratio of MC tomonomer nucleotide than ssDNAs of poly(C)20 and poly-(G)20. Between different cations, the formation of DNA-Ca2+-MC complex was found to be more stable and efficient thanDNA-Mg2+-MC complex due to a stronger Ca2+ bridge. Forlocalized MC release, DNA-Mg2+/Ca2+-MC complexes wereencapsulated in agarose gel, in which the release of MC lastedfrom a few days to more than 10 days, depending on variedDNA nanostructures. The released MC maintained its activityto inhibit pro-inflammatory response in macrophages. The

ideal dose and duration of MC release is dependent on specificclinical applications. For example, the treatment of chronicinflammation needs a stable release profile (near zero-orderrelease), which may prefer the release profile of poly(A)40-Mg2+-MC complex. On the other hand, for applications such asspinal cord injury, it requires an initial high-dose release forneuroprotection followed by low-dose release to target chronicinflammation.39 Therefore, poly(A)20-Mg2+-MC may be moresuitable for spinal cord repair.MC is a tetracycline analogue that has broad clinical

applications, due to its antibiotic, anti-inflammatory, andantiapoptotic activities. This study reported a metal ion-assisted interaction between MC and DNA structures thatincreased our understanding of biomolecular interactionsbetween DNA and small-molecule drugs. A similar effectmay also be observed for DNA binding with other tetracycline-like molecules,24 or metal-assisted assembly of DNA withchemotherapeutic drug doxorubicin.61 Compared with otherpolymers, DNA molecules have the unique advantages ofhighly tunable chemical composition, chain length, and self-assembled structures that can modulate their interaction withtherapeutic agents. In future developments, one could employthe power of switchable DNA nanostructures to develop ahighly tunable drug delivery system enabling versatile andcontrolled release of MC or other drug molecules. The self-assembled DNA-Mg2+-MC complexes could also potentially beused for anti-inflammatory and cytoprotective treatments inclinical applications, such as spinal cord therapy.39,43

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acsami.9b08126.

Detailed methods; absorption spectra for MC underdifferent Mg2+ concentrations (Figure S1); standard MCconcentration vs absorbance curve (Figure S2); test ofDNA molecules for inducing inflammatory macrophagesto release NO (Figure S3); ssDNA sequences used inexperiments (Table S1) and thermal annealing programfor preparing DNA nanostructures (Table S2); othercharacterizations (PDF)

■ AUTHOR INFORMATIONCorresponding Authors*(J.F.) E-mail: jinglin.fu@rutgers.edu.*(Y.Z.) E-mail: yz348@drexel.edu.ORCIDYonggang Ke: 0000-0003-1673-2153Jinglin Fu: 0000-0002-0814-0089Author Contributions#T.Z., J.N., and N.A. contributed equally to this work.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by an Army Research Office ECASEaward to J.F. (Grant W911NF1910240), DoD DURIP (GrantW911NF-16-1-0220), and the Cottrell College Science Awardto J.F. J.F. is also supported by the start-up funds from RutgersUniversityCamden. N.A. is sponsored by Saudi ArabianCultural Mission Scholarship. T.M. is sponsored by the REAP

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Program from Army Education Outreach. We are also gratefulto the Equipment Leasing Funds from the State of New Jersey.

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