Journal of Colloid and Interface Science 369 (2012) 453–459

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Journal of Colloid and Interface Science

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Efficiency of layered double hydroxide nanoparticle-mediated delivery of siRNAis determined by nucleotide sequence

Yunyi Wong a,1, Helen M. Cooper b,⇑, Kai Zhang b, Min Chen b, Perry Bartlett b, Zhi Ping Xu a,⇑a ARC Centre of Excellence for Functional Nanomaterials, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland,Brisbane, Queensland 4072, Australiab Queensland Brain Institute, The University of Queensland, Brisbane, Queensland 4072, Australia

a r t i c l e i n f o

Article history:Received 23 October 2011Accepted 15 December 2011Available online 23 December 2011

Keywords:Layered double hydroxide (LDH)siRNA deliveryCellular uptake efficiencyNucleotide sequenceNucleic acid–LDH interactions

0021-9797/$ - see front matter � 2012 Elsevier Inc. Adoi:10.1016/j.jcis.2011.12.046

⇑ Corresponding authors. Fax: +61 7 3346 6301 (H3973 (Z.P. Xu).

E-mail addresses: h.cooper@uq.edu.au (H.M. CoopeXu).

1 Present address: School of Chemical and Life SciencDover Road, Singapore 139651, Singapore.

a b s t r a c t

In this paper, we report the novel finding that the cellular delivery efficiency of siRNAs or their mimicdouble-stranded (ds)DNA using layered double hydroxide (LDH) nanoparticles is dependent upon thenucleotide sequence. Efficacy of LDH-mediated delivery of four different siRNAs into cortical neuronsand NIH 3T3 cells was found to vary widely (from 6 to 80%, and 2–11%, respectively). Our investigationinto the formation of dsDNA–LDH complexes through monitoring the dsDNA:LDH mass ratio at the pointof zero charge (PZC) indicated that the degree of intercalation of the individual dsDNA sequences into theLDH nanoparticles varied significantly. The dsDNA:LDH mass ratio at the PZC was found to be dependenton the nucleotide sequence. We further observed that PZC for each sequence was positively related to theextent of LDH-mediated internalization of the equivalent siRNA into neurons and fibroblasts. This novelfinding therefore suggests that the mass ratio at the PZC is a useful predictive tool with which to assessthe intercalation efficiency of selected siRNA sequences into the LDH interlayer and subsequent internal-ization into the cell cytoplasm. This finding will allow a more controlled approach to the design of suit-able siRNA sequences for LDH-mediated siRNA delivery.

� 2012 Elsevier Inc. All rights reserved.

1. Introduction

In recent years, layered double hydroxides (LDHs), also knownas anionic clay, have been shown to be an efficient delivery vehiclefor anionic drugs and nucleic acids [1–7]. Most LDH minerals con-form to the general formula [MII

x MIIIðOHÞ2þ2x�ðAn�Þ1=n �mH2O

(x = 1.5–4.0), where MII represents a divalent metal cation, MIII atrivalent metal cation and An� an anion [8,9]. Structurally, LDHscomprise alternating stacks of cationic brucite-like layers and hy-drated interlayer anions. In the brucite-like layer [MII

xþ1ðOHÞ2þ2x],the substitution of MII for MIII leads to a net positive charge([MII

x MIIIðOHÞ2þ2x�þ), which is neutralized by the interlayer hy-

drated exchangeable anions [(An�)1/n�mH2O], as exemplified bythe naturally existing hydrotalcite Mg3Al(OH)8(CO3)0.5�2H2O [8,9].This form of LDH (MgxAl(OH)2+2x(A)�mH2O, x = 1.5–4.0, A = Cl� orNO�3 , m = �2) is able to intercalate anionic biomolecules (such asDNA, RNA and anionic peptides) between the brucite-like layerswith high loading efficiency, providing protection against degrada-

ll rights reserved.

.M. Cooper), fax: +61 7 3346

r), gordonxu@uq.edu.au (Z.P.

es, Singapore Polytechnic, 500

tion [10,11]. In addition, the simple chemical composition (MgxAl–LDH) is highly biocompatible [4,12–14]. These properties have ledto the proposal that LDHs may act as an efficient drug/gene deliv-ery system. Indeed, in vitro studies have shown that they are effi-cient carriers of anionic drugs such as the anti-restenosis drug(heparin) [15,16], the anti-cancer drug (methotrexate) [17,18]and nucleic acids, especially small interfering RNAs (siRNAs)[3,6,11,12,18,19].

Efficient intercalation of organic anions (<1–2 nm in size) intothe LDH interlayer is normally achieved by heating to 90–100 �C[20–23]. This high-temperature exchange usually gives rise to anintercalation compound with good crystallinity, where the packingof the anions within the interlayer is dependent on the organicchain structure, negative charge density, and chain hydrophobicity[20–23]. As drug anions are physically constrained within the LDHinterlayers, they are protected from enzyme or oxidant attack.Therefore, the ability of LDHs to sequester and thus protect anionicdrugs is an advantageous property in the context of in vivo deliveryof biomolecules sensitive to oxidation (e.g. vitamin C) or nucleicacids susceptible to enzyme degradation [11]. Importantly, oncethe LDH–drug complex is internalized, the anionic drug can be re-leased from the interlayer into the cytoplasm in a controlled man-ner as a result of ‘reverse’ anion exchange with cytosolic chlorideor phosphate ions, thereby allowing sustained release over an ex-tended period of time [16].

454 Y. Wong et al. / Journal of Colloid and Interface Science 369 (2012) 453–459

Over recent years, the potential of LDH nanoparticles as a deliv-ery system for siRNAs and cDNAs encoding genes of therapeuticvalue has been demonstrated [3,6,11,12,18,19]. Desigaux et al. re-ported that linear double-stranded cDNAs between 100 and 8000base pairs (bps) in length can be intercalated into the LDH inter-layer via co-precipitation at 50–60 �C in the presence of excesscDNA [10]. In practice, such an optimized co-precipitation methodis not feasible for the intercalation of short double-stranded siRNAsequences (21–25 bp) due to their sensitivity to high temperature(above 60 �C), high salt concentrations (above 1–2 M) and alkalineenvironments (pH = 10–11). Therefore, if LDHs are to fulfill theirpotential as an siRNA-delivery system, less stringent intercalationprotocols need to be developed that yield high loading capacity.As a first step in achieving this goal, we have tested the loadingof siRNAs or mimic double-stranded DNAs (dsDNAs, Table 1) intothe LDH interlayer via anion exchange at 37 �C and investigatedthe structural determinants that promote efficient intercalationand cellular uptake.

We selected four functional siRNAs (21 bps) in this study. siR-NA-DCC#1 and siRNA-DCC#2 have been previously used in ourlaboratory to target the Deleted in Colorectal Cancer (DCC) gene[24], and siRNA–Htt#1 is able to effectively knock-down the mu-tated form of the human Huntington’s disease gene (Htt) [25]. Afourth sequence (siRNA-MAPK#1) targeting the Mitogen-ActivatedProtein Kinase (MAPK) mRNA was also chosen on the basis of itsguanine/cytosine (GC) content. To our knowledge, to date, therehas been no such comparative investigation into intercalation ofshort double-stranded nucleotides with different sequences. Wedemonstrate that efficient uptake is dependent on the nucleotidesequence. This finding enhances our understanding of siRNA/dsDNA–LDH interactions and illustrates that the interactions be-tween siRNA/dsDNA and LDHs are more complex than previouslythought. In addition, we present a predictive tool, based on thedsDNA:LDH mass ratio at the point of zero charge (PZC), to assesswhether the selected siRNA sequence will be efficiently interca-lated into the LDH interlayer.

2. Experimental

2.1. Preparation of LDH nanoparticles

Pristine MgAl–Cl LDH (with a designed formula Mg2Al-(OH)6Cl�2H2O) were prepared by aqueous co-precipitation in the

Table 1DNA:LDH mass ratio, zeta potential and average hydrodynamic particle size of dsDNA–LD

Name code DNA:LDH mass ratio Zeta (mV) a Avera

LDH 43.3 ± 2.0 108.0

dsDNA-Htt#1–LDH 1:10 36.3 ± 0.5 394.61:5 �2.5 ± 0.7 NAb

1:2 �34.4 ± 0.2 140.71:1 �36.7 ± 0.5 149.0

dsDNA-DCC#1–LDH 1:10 32.3 ± 0.6 743.31:5 �23.8 ± 0.4 187.21:2 �27.0 ± 0.3 134.61:1 �34.8 ± 2.2 129.3

dsDNA-DCC#2–LDH 1:10 40.1 ± 0.9 500.11:5 �2.4 ± 0.4 NAb

1:2 �29.9 ± 0.2 144.11:1 �38.2 ± 0.6 138.0

dsDNA-MAPK#1–LDH 1:10 �26.6 ± 0.7 259.51:5 �32.6 ± 0.3 157.41:2 �39.3 ± 0.2 128.81:1 �42.5 ± 0.4 134.0

a Data were means ± SEM; n = 3.b Particle size exceed measureable size limit of Nanosizer Nano ZS (6 lm).

c In the corresponding siRNA, base T was replaced with U.

presence of excess Mg2+ [26,27]. The salt solution containing3.0 mmol of MgCl2�6H2O (98%, Fluka) and 1.0 mmol of AlCl3�6H2O(98%, Fluka) in 10 mL of fresh MilliQ water was quickly added toa basic solution (40 mL) containing 6.0 mmol of NaOH (98%, Fluka)to precipitate under vigorous stirring at room temperature. Thesuspension was then stirred for 30 min. Care was taken to mini-mize CO2 uptake by purging N2 during preparation, precipitationand aging. The resulting white slurry was collected by centrifuga-tion at 5000g and washed twice with 40 mL fresh MilliQ water toremove excess salts. The wet cake was manually dispersed in Mil-liQ water (40 mL) and hydrothermally treated at 100 �C in a stain-less steel autoclave lined with Telfon for 16 h. The resultanttransparent suspension contained 4.0 mg/mL of homogenouslydispersed LDH nanoparticles. This suspension was normally di-luted four times (1.0 mg/mL of LDH) for intercalation ofoligonucleotides.

2.2. NIH 3T3 cell culture and uptake

Mouse fibroblasts (NIH 3T3s) were grown in DMEM medium(Gibco) supplemented with 10% fetal calf serum (JRH Biosciences),and penicillin (10 U/mL)/streptomycin (10 lg/mL) (Gibco), andmaintained at 37 �C and 10% CO2. NIH 3T3 cells were plated at aconcentration of 1.5 � 105 cells/well in Falcon 6-well plates. siR-NA–LDH complexes were prepared by mixing 6FAM-tagged siRNAwith pristine LDH at a siRNA:LDH mass ratio of 1:1 for 30 min at37 �C under gentle shaking. The resultant siRNA–LDH complexeswere added dropwise onto cells 24 h after plating, at a siRNA con-centration of 1.0 lg/mL. Controls included untreated NIH 3T3s orNIH 3T3s incubated with the equivalent amount of pristine LDHalone. After incubation for 4 h, the cells were collected and ana-lyzed using flow cytometry and light microscopy.

2.3. Cortical neuron cultures

Cortical neurons were dissected from mouse brains at embry-onic day 17.5 (E17.5) as previously described [6]. Neurons wereplated at a concentration of 5 � 105 cells/well in Falcon 6-wellplates. Twenty-four hours after plating, 1.0 lg/mL of siRNA–LDHwas added dropwise, and the neurons cultured for a further 4 h,after which internalization was assessed by flow cytometry.

H complexes.

ge size (nm) a Nucleotide sequencec GC%

± 3.0

± 33.4 dsDNA-Htt#1 85.7

± 3.2 Sense 50-GCGCCGCGAGTCGGCCCGAGG-30

± 2.9 Antisense 30-GCCGCGGCGCTCAGCCGGGCT-50

± 118.3 dsDNA-DCC#1 33.3± 24.0± 1.7 Sense 50-GCAATTTGCTCATCTCTAATT-30

± 1.4 Antisense 30-TTCGTTAAACGAGTAGAGATT-50

± 10.2 dsDNA-DCC#2 28.6

± 0.7 Sense 50-CGATGTATTACTTTCGAATTT-30

± 0.6 Antisense 30-GTGCTACATAATGAAAGCTTA-50

± 114.9 dsDNA-MAPK#1 33.3± 7.8± 1.1 Sense 50-GGGCTAAAGTATATCCATTTT-30

± 0.8 Antisense 30-CTCCCGATTTCATATAGGTAA-50

Y. Wong et al. / Journal of Colloid and Interface Science 369 (2012) 453–459 455

2.4. Flow cytometry

After incubation for 4 h, cells were washed once in PBS, de-tached using Trypsin–EDTA (Gibco) and then collected by centrifu-gation. The viability dye, 7-amino-actinomycin-D (7AAD) (20 lg/mL, Molecular Probes) was added to exclude dead and dying cells.Analysis was undertaken on a Becton Dickinson BDTM LSR II FlowCytometer (Becton Dickinson BD Biosciences) using 200 mW of488 nM laser light as the excitation source. Fluorescence emissionfrom 6FAM was collected through a 530/30 bandpass filter, and7AAD emission through a 660/40 bandpass filter. For each analysis,a population of 10,000 cells was counted, and the cell debris, dou-blets and non-viable cells were gated out using control cells (un-treated cells and untreated cells stained with 7AAD) as thereference. Untreated control cells were used as the baseline todemarcate the population of single viable cells that were 6FAM po-sitive. The percentages of single, viable cells that had taken up the6FAM–siRNA were quantified, and the histograms generated usingthe WEASEL software (Walter & Eliza Hall Institute, Australia). Sta-tistical significance was evaluated using unequal variance t-tests. Ap value of less than 0.05 was considered to be statistically signifi-cant. Data were presented as means ± SEM (standard error of themean).

2.5. Microscopic imaging

Uptake of 6FAM–siRNA–LDH complexes by NIH 3T3 cells orneurons was visualized using confocal microscopy. After 4 h incu-bation, cells were fixed in 4% paraformaldehyde (Sigma–Aldrich)for 10 min, washed 3�with PBS, incubated briefly with 40,6-diami-dino-2-phenylindole (DAPI) (1:11,000; Molecular Probes) and thenmounted onto Superfrost microscope slides (Menzel-Glaser) withDAKO fluorescent mounting media (DakoCytomation). Stainedcells were imaged on a Zeiss Axio Imager/Observer (Zeiss,Germany).

2.6. siRNA/dsDNA loading into LDH nanoparticles

To examine the formation of oligonucleotide–LDH complexes,the mimic dsDNAs (see Table 1 for sequences) were mixed withLDH nanoparticles in an aqueous suspension under gentle shakingon a platform shaker (1200 RPM) at 37 �C using a range ofdsDNA:LDH ratios. Typically, 1.0 lL of dsDNA solution (1.0 lg/lL)was mixed with 1–10 lL of diluted LDH suspension (1.0 lg/lL)and UltraPureTM DNase-free distilled water (Gibco) to a total vol-ume of 20 lL with shaking for 10 min at 37 �C. The particle sizedistribution and zeta potential were determined for the as-pre-pared dsDNA–LDH complexes in 1.0 mL solution (see Section 2.8).

2.7. Agarose gel electrophoresis

For gel electrophoresis analysis, 20 lL of dsDNA–LDH complexsuspension was loaded into each well of a 3% agarose gel (DNAgrade, Progen Industries Ltd.), which was then run in Tris BorateEDTA Buffer (TBE) containing ethidium bromide (Sigma) at 80 Vfor 40 min. dsDNA bands were visualized using a GelDoc UV illu-minator (Bio-rad Laboratories). To examine whether dsDNA associ-ated with LDH nanoparticles was protected, DNase I (0.2 lL of0.25 unit/lL DNase and 2.0 lL of buffer) was added to thedsDNA–LDH complex suspension and the control free dsDNA thatwas then digested at 37 �C for 1 h prior to electrophoresis.

2.8. Physicochemical characterization

Photon correlation spectroscopy (PCS) (Nanosizer Nano ZS,MALVERN Instruments) was used to analyze the particle size dis-

tribution and measure the zeta potential of LDH and siRNA/dsDNA–LDH nanoparticles. The X-ray diffraction pattern (XRD)was collected on a XRD Rigaku Miniflex Diffractometer (Rigaku Ja-pan) using Co Ka radiation (k = 0.17,902 nm) with a variable slitwidth in a step of 0.02� at a scanning speed of 2� per minute inthe range of 2h = 1.5–80�. In general, the LDH or dsDNA–LDH sus-pension was placed onto a quartz sample holder with the zerobackground and then air-dried to form a thin film for XRD patterncollection. Transmission electron microscopic images were re-corded on a JEOL JSM-1010 transmission electron microscope(TEM) (JEOL Ltd) at an acceleration voltage of 80 kV, and high-res-olution TEM images of LDH and dsDNA–LDH particles were ob-tained on a TECNAI F30 transmission electron microscope (FEICompany) at an acceleration voltage of 300 kV. Several drops ofLDH or dsDNA–LDH suspension were applied to a copper gridcoated with carbon film and air-dried before TEM analysis.

Elemental (Mg and Al) analysis was performed using induc-tively coupled plasma optical emission spectroscopy (ICPOES) ona Varian Vista Pro ICPOES instrument (Varian, Inc.). Chloride anal-ysis was done by colorimetric analysis on a SEAL AQ2 + DiscreetColorimetric Analyser (Ai Scientific). The carbon content of LDHsamples was measured with a CHNS-O analyser (FlashEA Series1112, Thermo Electron Co.).

3. Results and discussion

3.1. Characteristics of LDHs and oligonucleotide–LDH complexes

Pristine LDHs exhibited a zeta potential of 43.3 ± 2.0 mV and anaverage hydrodynamic diameter of 108.0 ± 3.0 nm (Z-average par-ticle size) with an approximate chemical formula of Mg1.9A-l(OH)5.8Cl0.8(CO3)0.1�1.5H2O. This average hydrodynamic diametermeasured using PCS was in good agreement with the expectedaverage lateral dimension of LDH nanoparticles (80–140 nm)shown in the TEM image (Fig. 1A). Fig. 1A also shows that the pris-tine LDH particles exhibited the characteristic hexagonal platelet-like morphology, as reported elsewhere [3,12,26,28].

The XRD pattern of the LDH sample (Fig. 1B(a)) displayed a ser-ies of sharp basal diffractions corresponding to planes (003), (006)and (009), indicating good crystallinity with the typical layeredstructure. The basal spacing (0.78 nm) was identical to that re-ported in the literature [8,9,26]. Based upon the full width at halfmaximum (FWHM) of the diffraction peak at the (003) planeand Scherrer’s Equation [29], the estimated thickness of the pris-tine LDH nanoparticles was calculated to be 13 nm with an averageaspect ratio of 8.

In some experiments, double-stranded DNA (dsDNA) wassubstituted for siRNA of the same sequence to avoid complicationsarising from RNAase-mediated degradation. When extreme care isnot taken, ubiquitous RNAses in the environment will degrade siR-NA, resulting in partial or complete loss of functional siRNA andconsequently inconsistent experimental observation. We have pre-viously shown that the hydrodynamic diameter and zeta potentialof the dsDNA–LDH and siRNA–LDH complexes were indistinguish-able [6], demonstrating that the physicochemical properties ofthese oligonucleotide–LDH complexes were equivalent at a massratio of 1:1. Based on the dimension of the dsDNA, intercalationof dsDNA into LDHs would be predicted to expand the LDH inter-layer spacing from 0.78 nm to 2.20–2.39 nm [1,10]. Contrary toexpectation, the XRD pattern generated by the dsDNA–LDH com-plexes (Fig. 1B(b)) indicated that this expansion did not occur.However, high-resolution TEM images revealed that a partialexpansion of interlayer spacing had occurred. As shown inFig. 1C, a TEM image of pristine LDH indicated a layered structurewith a spacing of 0.75 nm, which is close to that calculated from

200 nm

10 nm

(A) (B)

(D)(C)

10 nm

Fig. 1. (A) TEM image of LDHs, (B) XRD patterns of LDH (a) and dsDNA-Htt#1–LDH (b); high-resolution TEM images of LDH (C) and dsDNA-Htt#1–LDH (D), bars; interlayerwidth without dsDNA intercalation, arrow; expansion due to dsDNA intercalation.

456 Y. Wong et al. / Journal of Colloid and Interface Science 369 (2012) 453–459

the XRD pattern (0.78 nm). After intercalation of dsDNA-Htt#1(dsDNA:LDH mass ratio was 1:1), the interlayer spacing was seento expand to 2.20 nm (as estimated from the TEM image inFig. 1D, arrow). However, this expansion was infrequently seen(� one in 20 images) and only observed in restricted regions ofthe lattice (Fig. 1D, arrow). Nonetheless, the expansion of the inter-layer distance (2.2 nm) was comparable to that previously re-ported for intercalated nucleic acids (2.2–2.4 nm) [1,10]. Weinterpreted this regional expansion of the interlayer space to meanthat dsDNA-Htt#1 had only exchanged with a small proportion ofinterlayer Cl� anions, resulting in limited expansion of the LDHinterlayer spacing as could be observed in TEM images, but notby XRD.

3.2. Cellular uptake of siRNA nanoparticles

We have previously reported that LDH nanoparticles are able todeliver siRNA and dsDNA tagged with a 6FAM or FITC fluorophoreinto cultured mammalian cells and primary cultured neurons pre-dominantly via clathrin-mediated endocytosis [6,30,31]. An exam-ple is shown in Fig. 2. After incubation of NIH 3T3 fibroblasts withdsDNA-Htt#1–6FAM–LDH complexes for 4 h, strongly fluorescentpuncta can be seen within the cells (Fig. 2B, arrows). These punctahave previously been identified as early endocytic vesicles [6]. Dif-fuse 6FAM fluorescence was also seen throughout the cytoplasm,indicating release of dsDNA-Htt#1–6FAM from the endocytic com-partment into the cytoplasm (Fig. 2B). In contrast, no internaliza-tion was observed after incubation with dsDNA-Htt#1–6FAMalone (Fig. 2A). Importantly, we have also found that the extent

of internalization for dsDNA–LDH and siRNA–LDH complexes isequivalent [6].

To test the hypothesis that the oligonucleotide sequence influ-ences the efficiency of cellular uptake, we initially investigatedthe internalization of four distinct siRNA sequences (Table 1, siR-NA:LDH mass ratio of 1:1) by primary cortical neurons and NIH3T3s. Here, we exposed cells to the siRNA–6FAM–LDH complexesfor 4 h and then quantitated the percentage of 6FAM-positive cellsusing flow cytometry (fluorescence activated cell sorting; FACS).The cytoplasmic fluorescence of neurons and fibroblasts exposedto pristine LDHs or siRNA–6FAM alone was always equivalent tothe intrinsic background fluorescence displayed by the untreatedcells. Table 2 shows that 80.53% and 35.77% of neurons were6FAM-positive after incubation with the siRNA-Htt#1–6FAM–LDH and siRNA-DCC#2–6FAM–LDH, respectively, indicating amoderate to high efficiency of internalization. In striking contrast,the percentage of 6FAM-positive neurons seen after exposure tothe siRNA–6FAM–LDH complexes comprising the DCC#1 (6.40%6FAM-positive neurons) or MAPK#1 (5.63% 6FAM-positive neu-rons) sequences was greatly reduced. The uptake of the siRNA-Htt#1–6FAM–LDH and siRNA-DCC#2–6FAM–LDH complexes byNIH 3T3s was also more efficient compared to the uptake of theother siRNA–LDH complexes (Table 2). As previously reported,neuronal uptake of siRNA–LDH complexes was markedly more effi-cient than that observed for fibroblasts for all sequences tested [6].These data provide evidence that the dsDNA sequence has a pro-found effect on the ability of LDHs to transport siRNAs into mam-malian cells and suggest that the extent of intercalation into theLDH interlayer may be sequence-dependent.

Fig. 2. Internalization of siRNA–6FAM by NIH 3T3 cells after a 4 h exposure to siRNA–6FAM–LDH complexes. (A) Control: siRNA-Htt#1–6FAM alone, and (B) 1.0 lg/mL siRNA-Htt#1–6FAM–LDH complexes. siRNA-Htt#1–6FAM, green; DAPI, blue. Arrows indicate siRNA–6FAM–LDH complexes within endosomal vesicles. Scale bar: 16 lm. (Forinterpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 2Percentage of 6FAM-positive cells after 4 h exposure to 1 lg/mL LDH, siRNA–6FAM or siRNA–6FAM–LDH hybrids.

Neurons NIH 3T3 cells

Htt#1 DCC#1 DCC#2 MAPK#1 Htt#1 DCC#1 DCC#2 MAPK#1

Control 0.10 ± 0.10 0.03 ± 0.03 0.03 ± 0.03 0.23 ± 0.07 0.17 ± 0.07 0.53 ± 0.03 0.53 ± 0.03 0.20 ± 0.06LDH 0.13 ± 0.09 0.07 ± 0.03 0.07 ± 0.03 0.23 ± 0.03 0.20 ± 0.06 0.63 ± 0.12 0.63 ± 0.12 0.30 ± 0.06siRNA–6FAM 0.13 ± 0.03 0.03 ± 0.03 0.07 ± 0.03 0.17 ± 0.07 0.17 ± 0.07 0.47 ± 0.06 0.60 ± 0.00 0.17 ± 0.03LDH/siRNA–6FAM 80.53 ± 4.58a,b 6.40 ± 1.70a,b 35.77 ± 8.53a,b 5.63 ± 0.81a,b 11.03 ± 2.28a 2.73 ± 0.75a 9.50 ± 2.51a 1.80 ± 0.40a

a Unequal variance t-tests comparing siRNA–6FAM and siRNA–6FAM–LDH were significant for all siRNA–6FAM sequences (p < 0.05). Data were means ± SEM. n = 3.b Two-way Anova with Bonferroni’s post hoc test comparing siRNA–6FAM sequences was significant (p < 0.001). Data were means ± SEM. n = 3.

Y. Wong et al. / Journal of Colloid and Interface Science 369 (2012) 453–459 457

3.3. Complexation mode between dsDNA and LDH

We postulate that there are three possible modes of interactionbetween LDHs and siRNA/dsDNA: full intercalation, partial interca-lation, where the strand of siRNA/dsDNA protrudes from the inter-layer, and adsorption onto the LDH surface (Fig. 3). Similarinteraction modes have previously been reported in the contextof organic dye–LDH interactions, where they occur as a conse-quence of the anion exchange process. An investigation intoin situ kinetics demonstrated that during anion exchange the anio-nic dye (300–500 Dalton) interacted with the LDH via three dis-tinct modes, edge adsorption, external basal plane adsorptionand intercalation [32,33].

(A)

(B) Edge adsorption and Partial intercalation

(C)

Surface adsorption

Full intercalation

Fig. 3. Three interaction modes of siRNA/dsDNA–LDHs. (A) Adsorption onto theLDH basal plane, (B) edge adsorption and partial intercalation with part of siRNA/dsDNA protruding; (C) full intercalation.

In our system, surface adsorption (Mode A in Fig. 3) would beexpected to take place first between negatively charged siRNAs/dsDNAs and the positively charged LDH surface. This would alsoinvolve the surface anion exchange between siRNAs/dsDNAs andCl�, as observed in the dye intercalation [32,33]. Intercalation hasalso been clearly indicated in our TEM image (Fig. 1D). Althoughwe cannot determine from this image if the dsDNA is fully or par-tially intercalated (Mode C or B, respectively, Fig. 3), our previousstudies have demonstrated successful knock-down of target geneexpression [3,6]. This knock-down indicates that the siRNA withinsiRNA–LDH complexes is protected from enzymic degradation andimplies full intercalation of the dsDNA chain. Conversely, the pro-truding segment of partially intercalated dsDNA would be avail-able for cleavage as a result of partial intercalation.

To determine whether partial intercalation does occur underour intercalation conditions, we exposed the dsDNA–LDH com-plexes prepared at different mass ratios to DNAse I digestion, andthen subjected them to agarose gel electrophoresis and visualizedthe dsDNA using ethidium bromide. In the absence of DNase I (leftlanes in Fig. 4A), naked dsDNA-Htt#1 migrated quickly through thegel to the positive electrode. When dsDNA-Htt#1–LDH complexeswere prepared at mass ratios of 1:10 or 1:5, we observed a markeddecrease in the amount of free dsDNA at the bottom of the gel. In-stead, a large proportion of the dsDNA remained in the loadingwell, indicating that dsDNA-Htt#1 was immobilized due to inter-calation or surface adsorption by LDH nanoparticles. When thecomplexes were prepared at a mass ratio of 1:1 or 1:2, however,most of the dsDNA-Htt#1 migrated through the gel, indicating thatthe majority of the dsDNA was not associated with the LDH nano-particles. As expected, we observed complete loss of naked dsDNAafter exposure to the enzyme for all mass ratios (right lanes inFig. 4A). In contrast, a significant proportion of dsDNA was pro-tected from degradation at the mass ratios of 1:10 and 1:5 as indi-cated by the amount of dsDNA-Htt#1 retained in the loading wells.

(B)

(A)

Fig. 4. Agarose gel analysis of (A) dsDNA-Htt#1–LDH and (B) dsDNA–MAPK–LDHcomplex formation (anion exchange at 37 �C for 30 min) and lane 1: DNA ladder;lane 2: LDH only; lane 3: dsDNA alone (positive control); Lanes 4–7: dsDNA–LDHcomplexes at mass ratios from 1:10 to 1:1 as indicated; lane 9: LDH alone, lane 10:DNAse treated dsDNA alone; Lanes 11–14: DNAse treated dsDNA–LDH complexeswith the mass ratio from 1:10 to 1:1.

Fig. 5. Plot of zeta potential of dsDNA–LDH complexes against dsDNA:LDH massratio for four different dsDNAs.

458 Y. Wong et al. / Journal of Colloid and Interface Science 369 (2012) 453–459

However, the fluorescence intensity was lower than that observedin the absence of DNAse I (compare lane 4 with 11, and lane 5 with12 for the mass ratio of 1:10 and 1:5, respectively), indicating thatsome associated dsDNA had been degraded. Therefore, we con-clude that (1) a substantial proportion of immobilized dsDNAwas protected, confirming full intercalation of a proportion ofdsDNA-Htt#1 at mass ratios of 1:10 and 1:5; and (2) a fraction ofimmobilized dsDNA was degraded by DNAse I, indicating thatsome dsDNA-Htt#1 was partially intercalated and surface ad-sorbed in our system.

In the case of dsDNA–LDH complexes whose counterpart siR-NA–LDH complexes showed poor cellular uptake (Table 2), twobands corresponding to the free and immobilized dsDNA were alsoobserved in the agarose gels. However, the relative brightness ofthe free dsDNA bands at mass ratios of 1:10, 1:5 and 1:2 for thedsDNA-MAPK#1–LDH (Fig. 4B) and dsDNA-DCC#1–LDH (data notshown) was brighter than that observed for dsDNA-Htt#1–LDH(Fig. 4A). This indicates that more dsDNA-Htt#1 was immobilizedby LDH in the loading well compared with dsDNA-DCC#1 anddsDNA-MAPK#1, implying that the relative amount of these dsD-NAs associated with the LDH particles (surface adsorbed, fullyand/or partially intercalated) was lower than that for dsDNA-Htt#1. This finding is supported by the analysis of zeta potentialsfor the dsDNA–LDH complexes (Table 1).

3.4. dsDNA intercalation is sequence-dependent

To assess the extent of dsDNA intercalation for each sequence,we examined the zeta potentials of LDH complexes comprisingthe four dsDNA sequences over a range of mass ratios (Table 1).For the dsDNA-Htt#1–LDH complexes, we found that the zeta po-

tential decreased from 36.3 mV to �36.7 mV as the mass ratio in-creased from 1:10 to 1:1. Similarly, a larger negative zeta potentialwas correlated with an increase in the mass ratio for all dsDNA se-quences (Table 1, Fig. 5). Note that there was a marked divergencein the point of zero charge (PZC) (as determined from the curves inFig. 5) between the sequences efficiently internalized by neurons(Htt#1, DCC#2) and those that were poorly taken up (DCC#1,MAPK#1) (Table 2). The PZC for the dsDNA-Htt#1–LDH anddsDNA-DCC#2–LDH complexes occurred at the same mass ratioof 0.194 (1:5.2) lg/lg, whereas the PZC for dsDNA-DCC#1–LDHand dsDNA-MAPK#1–LDH complexes occurred at much lowermass ratios [0.156 (1:6.4) and 0.062 (1:16) lg/lg, respectively].

As the negative charge of the dsDNA will be offset once fullyintercalated (Mode C in Fig. 3), the decreasing zeta potential mustderive from the negative charge of dsDNA adsorbed onto the LDHsurface and protruding from the interlayers (Modes A and B inFig. 3). Thus, the decreasing zeta potential at the higher mass ratios(1:2 or 1:1) implies that a proportion of the dsDNA was surface ad-sorbed and/or partially intercalated per unit LDH mass. At the PZC,the exposed negative charge of the partially intercalated and sur-face adsorbed dsDNA must be equal to the positive charge carriedby LDH. Given that the structure and charge density of all dsDNAsand LDH are equivalent, one can assume that the total amount ofsurface adsorbed and partially intercalated dsDNA per unit LDHmass is the same for each sequence at the PZC:

LDHþ dsDNAðaqÞ ¼ LDH—dsDNAðadÞ:

These assumptions enable us to conclude that (1) the adsorp-tion equilibrium constant (K = [LDH–dsDNA(ad)]/[dsDNA(aq)] isthe same for all four dsDNAs; (2) the concentration of free dsDNA(e.g. dsDNA(aq)) at the equilibrium PZC will be approximately thesame for all four dsDNAs. Therefore, because we can exclude theamount of dsDNA remaining in the solution phase (dsDNA(aq))and adsorbed onto the surface (LDH–dsDAN(ad)) which will beequivalent for each sequence, a higher PZC mass ratio indicatesmore dsDNA is intercalated into the LDH interlayer. For example,the total contribution from protruding and surface adsorbeddsDNA would be between 0.043 and 0.065 lg (medium value is0.054) per lg of LDH as estimated in Ref. Note [34], thus the rela-tive amount of intercalated MAPK#1, DCC#1, HTT#1 and DCC#2would be 0.008, 0.104, 0.140 and 0.140 lg per lg of LDH, respec-tively. Thus, the mass ratio at which the PZC occurs provides ameasure of the relative extent of intercalation.

This proposed scenario is strongly supported by the cellularinternalization data presented in Table 2. Here, the relative extentof siRNA internalization by neurons and NIH 3T3 cells directly cor-

Y. Wong et al. / Journal of Colloid and Interface Science 369 (2012) 453–459 459

relates with the PZC mass ratio. We observed that the PZC occurredat a mass ratio of 0.194 for dsDNA-Htt#1 or dsDNA-DCC#2, a massratio markedly higher than that seen at the PZC for the dsDNA-DCC#1–LDH or dsDNA-MAPK#1–LDH complexes (Fig. 5). Basedon the above reasoning, the dsDNA-Htt#1 and dsDNA-DCC#2 com-plexes have a greater quantity of fully intercalated dsDNA, and thuswould be expected to transport dsDNA more efficiently into the cellcytoplasm. Therefore, we propose that the PZC mass ratio can beused as a predictive tool to assess whether the selected siRNA se-quence will be efficiently intercalated into the LDH interlayer.

Although all siRNAs/dsDNAs have the same charge distribution,we found significant differences in the zeta potential over a rangeof mass ratios as exemplified by the PZC for each dsDNA. Thisobservation, together with the different internalization efficiencyfor each siRNA (Table 2), provides strong evidence that the extentof intercalation is sequence-dependent. Examination of the se-quences of the four siRNAs/dsDNAs revealed that the most effi-ciently internalized siRNA-Htt#1 has a GC content of 86%,whereas the least efficient siRNA-DCC#1 and siRNA-MAPK#1 bothhave a GC content of only 33%, suggesting that the extent of inter-calation is proportional to the GC content. However, this postulatedoes not hold for siRNA-DCC#2, which also has a low GC content(29%), but exhibited an intermediate level of cellular internaliza-tion (Table 2). Therefore, the GC content per se does not appearto be the only parameter determining efficient intercalation. Weare now investigating the possibility that variation in secondarystructure may influence intercalation.

3.5. Why choose the mass ratio of 1:1 for cellular delivery?

It is true that at a mass ratio of 1:1 only a small proportion ofdsDNA-Htt#1 was associated with the LDH nanoparticles (Fig. 4),however, we nonetheless observed good cellular uptake at this ratio(Table 2). Our preference for using the 1:1 mass ratio for delivery ofsiRNA to mammalian cells is due to the following reasons. Firstly,the average hydrodynamic diameter of dsDNA–LDH complexes in-creased markedly (>200 nm) at ratios of 1:10 and 1:5 (Table 1) witha broader particle size distribution (indicated by larger SEM values),revealing that dsDNA induced complex agglomeration at these ra-tios. We attribute this to the transition of the zeta potential frompositive to negative (between �20 and 20 mV as indicated by thebroken lines in Fig. 5) that occurs at these lower ratios. In contrast,at the mass ratio of 1:1 the dsDNA–LDH complexes displayed only aslight increase in particle size (Table 1), indicating no significantagglomeration. Secondly, at the mass ratio of 1:1, the dsDNA load-ing per unit LDH mass was much higher, up to 0.3 lg(dsDNA)/lg(LDH), compared with the maximum 0.1 and 0.2 lg(dsDNA)/lg(LDH) in the mass ratios of 1:10 and 1:5, respectively, as has beenreported for plasmid DNA in our other studies [12].

4. Conclusions

In conclusion, we have reported for the first time that the nucle-otide sequence of siRNAs and short dsDNAs affects their interac-tion with LDH nanoparticles, and therefore determines theircellular uptake efficiency in cortical neurons and NIH 3T3 cells.Furthermore, we propose an intercalation model that explains, atleast in part, how different sequences may influence the interac-tion of dsDNAs/siRNAs with LDH nanoparticles.

Acknowledgments

This work was financially supported by the Australian ResearchCouncil (ARC) through Discovery Project Program (DP0879769)

and the ARC Centre of Excellence for Functional Nanomaterials.Dr. Xu gratefully acknowledges the award of ARC Australian Re-search Fellow (ARF). H.C. was supported by the Queensland StateGovernment Smart Futures Fellowship Scheme.

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sheets is about 13 nm, e.g. 16.7 layers, and thus the external surface is 100%/16.7 = 6% of the total surface (internal and external). This means that thesurface exchangeable anion is only 6% of the anion exchange capacity (AEC).Suppose the molecule weight of LDH and a nucleotide base is 250 and 330,respectively, then 50–75% exchange of surface anions (Cl� and CO2�

3 )withdsDNA will enable 1 lg of LDH to surface adsorb 0.04–0.06 lg of dsDNA.According to the report describing the formation of the positive zeta potentialof LDH-Cl (R.R. Delgado, M.A. Vidaurre, C.P.D. Pauli, M.A. Ulibarri, M.J. Avena,Surface-charging behavior of Zn–Cr layered double hydroxide. J. ColloidInterface Sci. 280 (2004) 431–441), loss of only 4–6% of the surface anionsresults in a 30–50 meV zeta potential. In the current case, further suppose 6–8% of the surface dsDNA is used to offset these lost anions to maintain thepoint of zero charge, then this implies that 1 lg of LDH can accommodateextra 0.003–0.005 lg of protruding dsDNA. Therefore, the total amount ofsurface adsorbed and protruding dsDNA per 1 lg of LDH is estimated to be0.043–0.065 lg, that is a mass ratio of 0.043–0.065 (lg/lg), with the mediumvalue of 0.054.