Home

Search

Collections

Journals

About

Contact us

My IOPscience

Anionic solid lipid nanoparticles supported on protamine/DNA complexes

This article has been downloaded from IOPscience. Please scroll down to see the full text article. 2008 Nanotechnology 19 285708 (http://iopscience.iop.org/0957-4484/19/28/285708) View the table of contents for this issue, or go to the journal homepage for more

Download details: IP Address: 150.214.36.4 The article was downloaded on 11/09/2013 at 09:23

Please note that terms and conditions apply.

IOP PUBLISHING Nanotechnology 19 (2008) 285708 (9pp)

NANOTECHNOLOGY doi:10.1088/0957-4484/19/28/285708

Anionic solid lipid nanoparticles supported on protamine/DNA complexes

Jiesheng Ye1 , Aihua Wang2 , Chunxi Liu1 , Zhijin Chen1 and Na Zhang1,3
1 School of Pharmaceutical Science, Shandong University, 44 Wenhua Xi Road, Jinan, Peoples Republic of China 2 Department of Respiratory Medicine, Afliated Qilu Hospital, Shandong University, 44 Wenhua Xi Road, Jinan, Peoples Republic of China

E-mail: zhangnancy9@sdu.edu.cn

Received 6 March 2008, in nal form 26 April 2008 Published 3 June 2008 Online at stacks.iop.org/Nano/19/285708 Abstract The objective of this study was to design novel anionic ternary nanoparticles for gene delivery. These ternary nanoparticles were equipped with protamine/DNA binary complexes (150200 nm) as the support, and the anionic formation was achieved by absorption of anionic solid lipid nanoparticles ( 20 nm) onto the surface of the binary complexes. The small solid lipid nanoparticles (SLNs) were prepared by a modied lm dispersionultrasonication method, and adsorption of the anionic SLNs onto the binary complexes was typically carried out in water via electrostatic interaction. The formulated ternary nanoparticles were found to be relatively uniform in size (257.7 10.6 nm) with a bumpy surface, and the surface charge inversion from 19.28 1.14 mV to 17.16 1.92 mV could be considered as evidence of the formation of the ternary nanoparticles. The uorescence intensity measurements from three batches of the ternary nanoparticles gave a mean adsorption efciency of 96.75 1.13%. Circular dichroism spectra analysis showed that the protamine/DNA complexes had been coated by small SLNs, and that the anionic ternary nanoparticles formed did not disturb the construction of the binary complexes. SYBR Green I analysis suggested that the ternary nanoparticles could protect the DNA from nuclease degradation, and cell viability assay results showed that they exhibit lower cytotoxicity to A549 cells compared with the binary complexes and lipofectamine. The transfection efciency of the ternary nanoparticles was better than that of naked DNA and the binary complexes, and almost equal to that of lipofectamine/DNA complexes, as revealed by inversion uorescence microscope observation. These results indicated that the anionic ternary nanoparticles could facilitate gene transfer in cultured cells, and might alleviate the drawbacks of the conventional cationic vector/DNA complexes for gene delivery in vivo. (Some gures in this article are in colour only in the electronic version)

1. Introduction
Gene therapy is becoming a promising approach in the treatment of disease as the understanding of the genetic basis of disease increases. The key technological impediment to successful gene therapy is vector optimization. Gene transfer vectors can be divided into two categories: viral and nonviral agents. Viral methods of gene delivery are efcient, but they suffer from several drawbacks, including a need
3 Author to whom any correspondence should be addressed.

for packaging cell lines [1], problems with safety, such as mutation [2, 3], carcinogenesis [4, 5], and the elicitation of an immune response that renders transgene expression transient [610]. In light of these concerns, non-viral gene transfer systems seem to be more applicable in that they are less toxic, less immunogenic, and easy to prepare. Non-viral gene transfer systems could be ideal systems for in vivo gene therapy [11]; meanwhile, they can be efcient for systemic delivery. Many non-viral plasmid DNA (pDNA) delivery systems have been developed, including cationic peptides [12],
1
2008 IOP Publishing Ltd Printed in the UK

0957-4484/08/285708+09$30.00

Nanotechnology 19 (2008) 285708

J Ye et al

polycationic polymers [13], and cationic lipids [14]. Solid lipid nanoparticles (SLNs) made from biodegradable solid lipids existing in the submicron size range have attracted increasing attention in recent years. SLNs may offer a number of technological advantages, such as the possibility of steam sterilization and lyophilization, large scale production with qualied production lines, and the use of substances that are generally accepted as safe [15]. However, this kind of cationic vector may not completely condense plasmid DNA; instead, half-condensed DNA complexes might form structures with a condensed core and a corona with protruding uncondensed DNA strands or other components [16]. Meanwhile, the interaction between cationic SLNs and plasmid DNA is difcult to control, and can lead to the formation of large aggregates. In addition, although cationic lipids have several advantages over other forms of nucleic acid transfer agents in cell culture and in vivo, cytotoxicity remains a problem, especially in vivo. For instance, cationic lipids cause several changes to cells, which include cell shrinking, reduced number of mitoses, and vacuolization of the cytoplasm [17]. Certain proteins such as protein kinase C may also be affected detrimentally by cationic amphiphiles [18]. To overcome these limitations as far as possible, a novel solid lipid nanoparticlegene vector which has small size, low toxicity, is stable and has high transfer ability is badly required. Over the past few years, in the context of nanoparticle engineering, considerable attention has been devoted to nanoparticles supported on surfaces of different nanocapsules. As a rule, a strong interaction between the support and these particles takes place, which makes them stable against coalescence. This approach has been generally applied to nanocapsules larger than 100 nm in diameter, which have a signicant afnity to the nanoparticles [19]. We are interested in designing anionic ternary nanoparticles composed of biocompatible lipids, such as Compritol ATO 888 and soya lecithin. There have been a few previous attempts to use anionic lipids for DNA transfer mediated by divalent or multivalent cations [2022]. Similarly, in the present work, anionic formation was achieved by anionic SLNs supported on the surface of protamine/DNA nanometric complexes. Protamines are highly charged, arginine-rich proteins that bind to DNA in a non-specic manner. There have been many reports demonstrating that the stability and transfection efciency of DNA are augmented by the addition of protamine sulfate [2325]. In addition, protamine contains four apparent nuclear localization signals (NLS) domains and increased nuclear localization of exogenous genes [24, 26]. In this paper, we describe the preparation and characterization of small, stable, and negatively charged DNA-loading ternary nanoparticles. The formulation was tested for physical parameters such as particle size, zeta potential, DNA-binding capacity, physical stability and in vitro release, and also for their biological properties such as cytotoxicity, resistance to nuclease degradation, and gene transfer efciency. 2

2. Experimental details
2.1. Amplication and purication of plasmid DNA (pEGFP- N1 ) pEGFP-N1 was used as reporter gene in this study. The pEGFP-N1 was amplied in the E coli DH5 bacterial strain and puried by using an EndoFree Plasmid Maxi Kit (Qiagen GmbH, Germany) according to the manufacturers instruction. The DNA purity was determined by agarose gel electrophoresis and by measuring the optical density (OD). Plasmid DNA with OD260 /OD280 1.83 was collected. 2.2. Preparation of protamine/DNA binary complexes For the condensation of pDNA, 0.25 ml of 0.2 mg ml1 aqueous solution of pDNA was added dropwise to 1.25 ml of protamine sulfate solutions (various mass concentration) under gentle vortexing for 20 s. Then the sample was incubated at room temperature for 30 min to facilitate complexation [27]. 2.3. Agarose gel electrophoresis The complexes was subjected to a 1% (w/v) agarose gel electrophoresis for 40 min at 85 V cm1 . Images were obtained using a UV transilluminator and a digital imaging system (IS-2200, Alpha Innotech, USA). 2.4. Engineering of anionic SLNs supported on the surface of protamine/DNA complexes Blank small SLNs were prepared by a modied lm dispersionultrasonication method [28]. Briey, stearic acid (100 mg) and lecithin (300 mg) were dissolved in 5 ml chloroform, and the solution was evaporated at 45 C until a thin layer uniform lm was formed at the bottom of the ask. The residue of the organic solvent was expelled under vacuum over night. Then 5 ml of aqueous phase containing surfactant S-40 was added to allow the lm to expand and disperse, and the mixture was further dispersed ultrasonically for 5 min. After that, the suspension was poured into cold water under stirring at 1000 rpm (RW 20.n, Kika Labortechnik, Germany) for 4 h at 2 C under an ice bath to allow the hardening of SLNs. The SLNs were obtained by centrifuging at 17 000 rpm for 1 h at 4 C (Shanghai Anting Scientic Instrument Co., Ltd, China), washed twice, subsequently resuspended in Milli-Q water, and ltered through a membrane with 0.45 m pore size (Phenomenex, 25 mm lter, CA, USA). Adsorption of anionic SLNs onto the surface of the protamine/DNA binary complexes was typically carried out in Milli-Q water for 30 min at room temperature. The adsorption procedure was as follows. 100 l of SLNs (about 1.2 mg ml1 lipid) was added to 100 l of binary complex suspension at various protamine concentrations under stirring for 30 min. The resulting ternary nanoparticles were centrifuged, washed and resuspended in phosphate buffered saline (PBS, pH 7.4), and the supernatant was collected and used for quantication of the adsorption efciency. The preparation protocol for ternary nanoparticles is shown in gure 1.

Nanotechnology 19 (2008) 285708

J Ye et al

Germany) in a thermostated holder. To improve the signal-tonoise ratio, three scans were accumulated and averaged. After running a blank spectrum with PBS, aliquots of protamine solution, binary complexes, three kinds of ternary nanoparticle (composed of 0.3 mg ml1 lipid, 0.6 mg ml1 lipid and 1.2 mg ml1 lipid, respectively) were added to the cuvette, respectively. All experiments were conducted in triplicate. 2.8. Exposure of ternary nanoparticles to DNase I To test the protection effect of the plasmid DNA from nuclease digestion, DNase I mediated digestion was evaluated using agarose gel electrophoresis. Binary complexes, ternary nanoparticles and free DNA (1 g) were separately incubated with DNase I (2.5 U) in DNase/Mg2+ digestion buffer (50 mM, Tris-Cl, pH 7.6, and 10 mM MgCl2 ) for various times at 37 C and with agitation at 700 rpm. The enzymatic digestion was terminated with 1 M EDTA solution. The samples were transferred into a 10 ml centrifuge tube followed by addition of appropriate amount of methanol/chloroform/isoamyl alcohol (14:25:1) mixture, vortexed vigorously for 10 s, and centrifuged at 5000 rpm for 10 min at 4 C. The top aqueous phase was carefully removed and transferred to a new tube, then 0.1 volume of 3 M sodium acetate and 2 volumes of ice cold absolute ethanol was added, mixed by vortexing and placed in 20 C overnight, then centrifuged at 12 000 rpm for 2 min at 4 C. DNA pellets were resuspended in TrisEDTA buffer (TE: 10 mmol l1 Tris-HCl, 1 mmol l1 EDTA, pH 8.0). Samples were analyzed by the SYBR Green I assay, as described above. 2.9. In vitro release determination In vitro release studies were performed in phosphate buffer (pH 7.4). An aliquot of binary complexes and ternary nanoparticles (equivalent to 1 g DNA) was placed in a polypropylene centrifuge tube containing 1 ml of PBS and vortexed. The release percentage for each sample was determined only once at a selected time point. The tubes were then placed in a 37 C shaking water bath at 100 rpm and aliquots (0.5 ml) were withdrawn at each time interval. The DNA concentrations were determined using the SYBR Green I assay method mentioned above. 2.10. Cell viability studies In order to investigate the biocompatibility of ternary nanoparticles and determine the safe dose for the further experiments of this study, an in vitro evaluation experiment was performed using the A549 cell line. The cells were seeded into a 24-well plate at a density of 2 105 cells per well in 0.2 ml of RPMI1640 with 10% FBS and antibiotics, and then cultured at 37 C for 24 h. After culture, the growth medium was removed and the cells were immediately treated with various amounts of ternary nanoparticles for another 48 h. The effect of different treatments on cell viability was assessed by a CCK8 assay kit according to the manufacturers procedures, and the absorbance at 450 nm was measured using a microplate reader (Model 680, BIO-RAD, USA). Untreated cells were taken as 3

Figure 1. Schematic illustration of the structure of anionic ternary nanoparticles.

2.5. Physicochemical properties of ternary nanoparticles The morphology of ternary nanoparticles was examined by transmission electronic microscopy (JEM-1200EX, Japan). Samples were prepared by placing a drop of the ternary nanoparticles resuspension onto a copper grid and airdrying, following negative staining with one drop of 2% aqueous solution of sodium phosphotungstate for contrast enhancement. The air-dried samples were then directly examined under the transmission microscope. The size and zeta potential of the particles were analyzed in triplicate by photon correlation spectroscopy and laser Doppler anemometry, respectively, using a particle sizer (Zetasizer 3000 HAS, Malvern Instruments Ltd, Malvern, Worcestershire, UK). 2.6. Determination of the adsorption efciency The entrapment efciency of the ternary nanoparticles was determined using the SYBR Green I uorometry method [29]. The concentration of plasmid was assessed with a uorescence spectrophotometer (Hitachi 850, Japan) dyed with SYBR Green I according to the directions of the SYBR Green I kit after incubation with the decondensing agent. In detail, the complexes in the supernatant were decomposed with 4 M NaCl, and the concentration of DNA was determined by uorescence, comparing with the supernatant from blank SLNs. The amount of DNA loaded in the ternary nanoparticles was calculated according to the linear calibration curve of DNA. 2.7. Circular dichroism studies The formation of ternary nanoparticles and conformational change of protamine were analyzed by circular dichroism (CD). CD spectra were collected from 280 to 180 nm in 1 nm increments at 20 C using a Chirascan CD spectrometer (Applied Photophysics, Leatherhead, UK) equipped with a 0.1 mm path-length quartz cuvette (106-QS, Hellma,

Nanotechnology 19 (2008) 285708

J Ye et al

Figure 2. Gel retarding analysis of protamine/DNA complexes.

Figure 3. The effects of anionic SLNs on the decondensation of protamine/DNA complexes. Protamine sulfate/DNA complexes were of the following weight ratios: 0:1 in lane 1; 2:1 in lanes 2 and 3; 4:1 in lanes 4 and 5; 8:1 in lanes 6 and 7; 16:1 in lanes 8 and 9. The complexes in lanes 3, 5, 7, 9 were incubated with 10 l anionic SLNs (about 1.2 mg ml1 lipid). 200 ng DNA was loaded into each well.

control with 100% viability, and cells without the addition of CCK-8 were used as a blank to calibrate the spectrophotometer to zero absorbance. The relative cell viability (%) compared to control cells was calculated by [Abssample /Abscontrol ] 100. 2.11. In vitro transfection assay The A549 cells were seeded into 24-well plates at a density of 2 105 cells/well in 1 ml of growth medium, 24 h prior to transfection. When the cells were at about 80% conuence, the culture medium was replaced with 200 l transfection medium (serum-free) containing a denite volume of samples (50 l of protamine/DNA complexes or 50 l of ternary nanoparticles). Lipofectamine was used as a positive control, and the formulation of lipofectamine/DNA complexes carried out according to the manufacturers protocol. Naked DNA was used as a negative control. After incubation for 6 h at 37 C in a 5% CO2 incubator, the cells received 1 ml of complete medium and were incubated sequentially until 48 h post transfection.

3.2. Engineering of anionic SLNs supported on the surface of protamine/DNA complexes The small SLNs were obtained by the lm dispersion ultrasonication process in the presence of an excess of nonionic surfactant S-40. Despite washing the particles by two successive centrifugation/redispersion steps, still a little residual S-40 was evidenced. A similar result has been reported for PLGA nanoparticles stabilized by F68 [31]. Nevertheless, the surface potential of these obtained nanoparticles remained in the 30 to 40 mV range at low ionic strength. Anionic SLNs supported on the surface of protamine/DNA complexes were expected to occur via attractive electrostatic interactions between the negatively charged SLNs and the cationic protamine/DNA complexes. In preliminary trials, we found that anionic SLNs could decondense DNA from the protamine/DNA complexes at high concentration. Hence, the effect of weight concentration ratio of anionic SLNs to protamine on the decondensing of protamine/DNA complexes was investigated rst of all. Obviously, anionic SLNs would decondense DNA at approximately 3.75-fold lipid weight excess to the protamine weight; that is, the stable ternary nanoparticles could be formed only when the weight ratio of SLNs to protamine was below this dividing line (gure 3). In a preliminary study, the effects of phosphate concentrations of PBS on condensation of binary complexes were investigated, and the ability of anionic SLNs supported on the surface of protamine/DNA complexes had been found to be dependent on the phosphate concentration (data not shown). The lower the phosphate concentration, the more easily adsorption of SLNs on the binary complexes could develop. A similar result has been reported by another group which indicated that the ternary nanoparticles could be assembled successfully in salt-free water [32]. 3.3. Physicochemical properties of ternary nanoparticles Transmission electron microscopy (TEM) was conducted to investigate the morphology of the ternary nanoparticles. This technique was selected to conrm the formation of the ternary nanoparticles. The TEM pictures showed that anionic SLNs had spherical or ellipsoidal shapes (gure 4(A)), while the binary complexes displayed irregular shapes: a corona 4

3. Results and discussion

3.1. Condensation of DNA by protamine The concentration of the protamine (Cprotamine ) at various charge ratios (+/) was calculated using the following equation: Charge ratio

= {(Cprotamine n cation )/MWprotamine }/(CDNA /MWDNA )

where n cation denotes the number of arginine residues in protamine. MWprotamine and MWDNA represent the molecular weight of the protamine (average: 4250) and one nucleotide (average: 308), respectively. CDNA denotes the concentration of pDNA (0.2 mg ml1 ). Figure 2 shows the gel retardation results of the complexes with nitrogen/phosphate (N/P) ratio from 0.25 to 6.0. The complete retardation of the complexes can be seen when the N/P ratio was above 1.5, which suggests that protamine and plasmid DNA started to form tight complexes with redundant positive surface charge. A similar result has been reported by another group [30].

Nanotechnology 19 (2008) 285708

J Ye et al

Figure 4. Transmission electron micrographs of blank anionic SLNs (A), binary complexes (B), and ternary nanoparticles (C).

Table 1. Particle size, polydispersity index, and zeta potential of binary complexes and ternary nanoparticles (n = 3). Sample Size (nm) PDI Zeta potential (mV)
Circular Dichroism (mdeg)

1.5

Anionic SLNs 19.2 1.5 0.15 0.01 35.17 2.72 Binary complexes 165 28.1 0.13 0.02 19.28 1.14 Ternary nanoparticles 257.7 10.6 0.21 0.05 17.16 1.92

0.5

structure with protruding uncondensed DNA strands or peptide chains of protamine is shown in gure 4(B). As anionic SLNs were added to binary complexes, anionic SLNs (white in color) were adsorbed onto the surface of the binary complexes (black in color) to form the round ternary nanoparticles. Interestingly, the ternary nanoparticles presented a bumpy surface (see gure 4(C)); the formation of this shape can be explained by the fact that the protruding chains of components were recondensed by anionic SLNs via electrostatic interaction, but the anionic SLNs could not fuse with the protruding chains of the support as liposomes, because of the solid shell of the anionic SLNs. The mean particle diameters, polydispersity index, and zeta potential determined by particle sizer before and after adsorption are reported. As shown in table 1, the ternary nanoparticles mean sizes increased about 56% with respect to the binary complexes; however, the particle size of the ternary nanoparticles (257.7 10.6 nm) was larger than the sum total of the binary complexes (165 28.1 nm) and small SLNs (19.2 5.5 nm). It was very likely that the protruding chains of components lay between the small SLNs and the inner core of binary complexes, and, as seen in gure 4(C), a toroidal interlayer formed. In addition, as expected, the results showed that the ternary nanoparticles were negatively charged; thus, adsorption of anionic SLNs onto binary complexes was further attested by the surface charge inversion. SYBR Green I is a sensitive dsDNA stain for quantitating dsDNA in solution. A model of protamineDNA binding holds that protamines convert DNA molecules into a very compact state [33]; the complexes possess a low binding capacity for several dyes and uorochromes [34], such as methyl green, ethidium bromide, and SYBR Green I. Therefore, the complexes were treated with a high concentration of salt (4 M 5

-0.5

-1

-1.5 180

190

200

210

220

230

240

250

260

270

280

Wavelength (nm)

Figure 5. CD spectra of protamine in PBS (a), binary complexes (b), ternary nanoparticles (composed of 0.3 mg ml1 lipid (c), 0.6 mg ml1 lipid (d), and 1.2 mg ml1 lipid (e)), pH 7.4, at 20 C.

NaCl) to release free DNA. The results of the SYBR Green I assay from three batches of the ternary nanoparticles gave a mean adsorption efciency of 96.75 1.13%. 3.4. Circular dichroism studies Circular dichroism (CD) is an excellent method for the study of the conformations adopted by proteins in solution. CD measurements have two major advantages: they can be made on small amounts of material in physiological buffers, and they provide one of the best methods for monitoring any structural alterations that might result from changes in environmental conditions, such as pH, temperature, and ionic strength [35]. CD spectra of the protamine demonstrated a negative peak at 195 nm (gure 5(a)); the most important contributor here may be the peptide bond and the histidine residues in the amino acid side chains of salmine [36]. The addition of DNA to the protamine did not produce any change in the overall shape of the spectrum, although the intensity was affected (gure 5(b)), while the addition of anionic SLNs resulted in

Nanotechnology 19 (2008) 285708

J Ye et al

Activity of DNA(%)

naked DNA binary complex ternary nanoparticle

Figure 7. Agarose gel electrophoresis of pDNA extracted from different vectors after being incubated with DNase. Lane 1: control pDNA; lane 2: naked pDNA digested for 30 min; lane 3: binary complexes digested for 1 h; lane 4: ternary nanoparticles digested for 1 h; lane 5: binary complexes digested for 4 h; lane 6: ternary nanoparticles digested for 4 h. The lower band is supercoiled (S.C.) pDNA and the upper band is open circular (O.C.) pDNA.

Digestion time(min)

Figure 6. Binary complexes, ternary nanoparticles and naked DNA control digested with DNase I at different times (n = 3).

the formation of anionic ternary nanoparticles. Meanwhile, as the SLN concentration increased, the ellipticity at 195 nm decreased obviously, though the shape of the spectrum still remained the same as that of the binary complexes (gures 5(c) and (d)). No evident change in the overall shape of the signal due to the peptide bond and the histidine residues of protamine in these systems were not affected. The maximum decrease at 195 nm, which was about 20% of the initial ellipticity, occurred at high SLN concentration of 1.2 mg ml1 lipid (gure 5(e)). Thus, the result has shown that the exposed protamine in DNA/protamine complexes was covered by anionic SLNs; furthermore, even higher levels of anionic SLNs did not produce obvious conformational transitions and NLS-like sequence change of protamine. 3.5. Exposure of ternary nanoparticles to DNase I In order to test whether ternary nanoparticles could protect pDNA from nuclease digestion, ternary nanoparticles supported on protamine/DNA complexes were exposed to DNase I. The open circular and supercoiled forms of pDNA are reported to be similar in their ability to transfect cells [37], so the ability of ternary nanoparticles and binary complexes protecting DNA against degradation of DNase I could be quantitated by SYBR Green I assay, and the results are shown in gure 6. After 4 h digestion, the protection from enzymatic degradation with ternary nanoparticles and binary complexes showed averages over 85% and 60% of total DNA recovery. The naked DNA control was completely fragmented within 30 min. Degradation of DNA by endonucleases, such as DNase I, is a major barrier for gene delivery in vitro and in vivo [38]. Our studies showed that ternary nanoparticles could protect DNA against degradation by DNase I more preferably compared with binary complexes. The structural and functional integrity of pDNA is shown in gure 7. The control pDNA appeared almost exclusively as the supercoiled conformation (lane 1). Over 80% supercoiled pDNA was observed after being incubated with DNase for 1 h and extraction from binary complexes and 6

ternary nanoparticles (lanes 34). After 4 h digestion, the pDNA remained a large extent supercoiled pDNA extracted from ternary nanoparticles in spite of a small amount of open circular pDNA also presenting (lane 6); however, no more than 60% supercoiled pDNA was observed from binary complexes (lane 5), which meant that the ternary nanoparticles obviously increased the stability of pDNA compared with binary complexes. 3.6. In vitro release determination The in vitro release studies of DNA from binary complexes and ternary nanoparticles were carried out in phosphate buffer (pH 7.4) at 37 C (gure 8). The release of DNA from ternary nanoparticles followed the RitgerPeppas equation and could be modeled by the following equation: ln R = 0.6038 ln t + 1.5292, r = 0.9942. The release of DNA from binary complexes was tted to the Higuchi equation and could be expressed by the following equation: R = 8.2419t 1/2 0.6640, r = 0.9979 (table 2). The release prole of DNA from ternary nanoparticles was triphasic. The initial fast release of around 23% of the DNA from ternary nanoparticles was observed in the rst 12 h, which could be explained by DNA desorption from the outer surface of the binary complexes. Subsequently, the release of DNA exhibited sustained-release property, and the accumulated DNA release percentage at 48 h was about 40%; the slow release of the DNA in this stage was attributed to the fact that small size solid lipid nanoparticles adsorbed on the outer surface of binary complexes hindered the release of DNA in the core of binary complexes. After 2 days, the release of DNA from ternary nanoparticles was 76.3% within the monitored period of 96 h. With deuxion and degradation of the adsorbed solid lipid nanoparticles, the rate of DNA release was relatively fast, and it displayed a similar prole as that of the binary complexes. 3.7. Cell viability studies Toxicity of gene vectors including viral vectors, cationic liposomes, and polymeric cations is a major barrier to efcient delivery of exogenous genes [39]. In our study, the exponentially grown A549 cells were treated with binary complexes and ternary nanoparticles, and the cell viability was measured using a CCK-8 assay kit.

Nanotechnology 19 (2008) 285708

J Ye et al

Cumulative release (%)

ternary nanoparticle binary complex

Time (h)

Figure 8. In vitro release prole of DNA from binary complexes and ternary complexes in PBS at 37 C (n = 3). Table 2. The regression equation of binary complexes and ternary nanoparticles release in vitro. (Note: R % = the rate of released drug at different time in the equation.) Formulation Binary complex Equation type Zero-order First-order Higuchi RitgerPeppas Zero-order First-order Higuchi RitgerPeppas Equation

k
0.7656 0.0155 8.2419 0.5776 0.7217 0.0133 7.5974 0.6038

C
13.08 4.5067 0.6640 1.8494 8.1536 4.562 4.1877 1.5292

r
0.9674 0.9942 0.9979 0.9886 0.9830 0.9827 0.9872 0.9942

Ternary nanoparticles

R = kt + C ln(100 R ) = kt + C R = kt 1/2 + C ln R = k ln t + C R = kt + C ln(100 R ) = kt + C R = kt 1/2 + C ln R = k ln t + C

Figure 9. Cell viabilities of lipofectamine, binary complexes, ternary nanoparticles and control cells in A549 cell line (n = 3). p < 0.05 compared with lipofectamine and binary complexes. Students t test was used to determine the level of signicance at p < 0.05.

The major factor affecting the cytotoxicity of cationic nonviral vectors was the aggregation of cationic vectors onto cell surfaces, which impaired the important membrane functions of cells. Especially, electrostatic interaction between cationic charged groups of the cationic vectors, such as cationic lipid, and negatively charged surface groups of the cell might disrupt the membrane functions of cells [40]. The overall electrical potential of the ternary nanoparticles prepared by our method was negative; thus ternary nanoparticles might overcome the disadvantages of cationic non-viral vectors induced by excessive positive charge. The ternary nanoparticle formulations were found to be pretty low in toxicity, as the average cell viabilities were between 90 and 100% compared with control cells. 3.8. In vitro transfection investigation In order to evaluate the enhancing effect of ternary nanoparticles on transferring DNA in vitro, A549 cells were used to investigate the transfection efciencies. After transfection for 24 h, the pictures from the inversion uorescence microscope which collected the green uorescence sent out by the transfected cells indicated that the cells incubated with the ternary nanoparticles successfully expressed enhanced green uorescent protein. The results indicated that the ternary nanoparti7

Lipofectamine, which is commonly used in in vitro gene transfection, was also evaluated for comparison, and the results showed that lipofectamine exhibited higher cytotoxicity than ternary nanoparticles at the test concentrations. Furthermore, compared with binary complexes, it was demonstrated that ternary nanoparticles exhibit a low cytotoxicity to A549 cells within the monitored period of 48 h ( P < 0.05, gure 9).

Nanotechnology 19 (2008) 285708

J Ye et al

anionic ternary nanoparticles might possess potential value in delivering pDNA to target cells in vivo.

Acknowledgments
The work was supported by the National Natural Science Foundation of China, No. 30572267. We gratefully acknowledge Dr Xiuzhen Wu from Institute of Natural Product Chemistry, Shandong University, for the inversion uorescence micrographs.

References
[1] Lollo C P, Banaszczyk M G and Chiou H C 2000 Curr. Opin. Mol. Ther. 2 13642 [2] Marshall E 1995 Science 269 10505 [3] Romano G 2005 Drug News Perspect. 18 3116 [4] Powell S K, Kaloss M, Burimski I, Weaver L, Long Z, Lyons R, McGarrity G J and Otto E 1999 Hum. Gene Ther. 10 212332 [5] Check E 2003 Nature 421 678 [6] Freimuth P 1996 J. Virol. 70 40815 [7] Lefesvre P, Attema J, Lemckert A, Havenga M and van Bekkum D 2003 BMC Mol. Biol. 4 418 [8] Chen D, Murphy B, Sung R and Bromberg J S 2003 Gene Therapy 10 9918 [9] Jooss K and Chirmule N 2003 Gene Therapy 10 95563 [10] Zaiss A K, Liu Q, Bowen G P, Wong N C, Bartlett J S and Muruve D A 2002 J. Virol. 76 458090 [11] Makiya N and Mitsuru H 2002 Biol. Pharm. Bull. 25 27583 [12] Fabre J W and Collins L 2006 Curr. Gene Therapy 6 45980 [13] De Smedt S C, Demeester J and Hennink W E 2000 Pharmacol. Res. 17 11326 [14] Clark M and Hersh E M 1999 Curr. Opin. Mol. Ther. 1 15876 [15] Tabatt K, Sameti M, Olbrich C, M uller R H and Lehr C M 2004 Eur. J. Pharmaceut. Biopharmaceut. 57 15562 [16] Budker V, Trubetskoy V and Wolff J A 2006 Biopolymers 83 64657 [17] Lappalainen K, Jaaskelainen I, Syrjanen K, Urtti A and Syrjanen S 1994 Pharmacol. Res. 11 112731 [18] Aberle A M, Tablin F, Walker N J, Gruenert D C and Nantz M H 1998 Biochemistry 37 653340 [19] Gubin S P and Korobov M S 2004 NANO-2004 India Keynote and Invited Speakers NANO India International Committee [20] Liang H, Harries D and Wong G C 2005 Proc. Natl Acad. Sci. USA 102 111738 [21] Patil S D, Rhodes D G and Burgess D J 2005 Biochim. Biophys. Acta 1711 111 [22] Cheng H, Zhang K, Libera J A, Olvera de la Cruz M and Bedzyk M J 2006 Biophys. J. 90 116474 [23] Balhorn R, Brewer L and Corzett M 2000 Mol. Reprod. Dev. 56 2304 [24] Gao X and Huang L 1996 Biochemistry 35 102736 [25] Li S and Huang L 1997 Gene Therapy 4 891900 [26] Ziemienowicz A, Gorlich D, Lanka E, Hohn B and Rossi L 1999 Proc. Natl Acad. Sci. 96 372933 [27] Ye J S, Zhang N, Ma C H, Huang G H and Luan F 2007 Chin. Pharm. J. 42 16448 [28] Lu B, Xiong S B, Yang H, Yin X D and Chao R B 2006 Eur. J. Pharm. Sci. 28 8695 [29] Ye J S, Zhang N, Ma C H and Huang G H 2007 Chin. J. Pharm. Anal. 27 176972 [30] Masuda T, Akita H and Harashima H 2005 FEBS Lett. 579 21438 [31] Trimaille T, Pichot C, Elaissari A, Fessi H, Briancon S and Delair T 2003 Colloid Polym. Sci. 281 118490

Figure 10. Fluorescent microscopy of A549 cells transfected by plasmid encoding enhanced green uorescence protein (EGFP) with different carriers. Gene expression was examined after 24 h and 48 h post transfection, respectively.

cles with a diameter less than 0.3 m used in this study would be suitable for the uptake of A549 cells. It was found that the transfection efciency of ternary nanoparticles was higher than that of protamine/DNA binary complexes in A549 cells (gures 10(E) and (G),10(F) and (H)); it was possible that the cytotoxicity of the binary complexes impacted the phagocytosis ability of the cells, which was in accordance with the result of the cell viability study. As shown in gures 10(C) and (G), the transfection efciency of ternary nanoparticles was lower than that of lipofectamine/DNA complexes at 24 h, while a similar transfection efciency was obtained at 48 h (gures 10(D) and (H)), which was attributed to the fact that DNA could only be released slowly from the ternary nanoparticles through dissolution and diffusion; these results are consistent with the in vitro release prole of ternary nanoparticles.

4. Conclusion
We have reported novel anionic ternary nanoparticles which were composed of anionic solid lipid nanoparticles supported on protamine/DNA complexes as non-viral vectors for gene delivery, assembled via electrostatic interaction. The resulting ternary nanoparticles had small size and good colloidal stability. Compared with protamine/DNA binary complexes, the ternary nanoparticles exhibited lower cytotoxicity and better protection function from nuclease degradation. The transfection efciency of ternary nanoparticles was better than that of naked DNA and binary complexes, and almost equal to that of lipofectamine/DNA complexes revealed by inversion uorescence microscopy. Further efforts are warranted to incorporate targeting moieties for increased cell uptake and nuclear translocation. The results of this study suggest that 8

Nanotechnology 19 (2008) 285708

J Ye et al

[32] Junghans M, Kreuter J and Zimmer A 2001 Biochim. Biophys. Acta 1544 17788 [33] Porschke D 1991 J. Mol. Biol. 222 42333 [34] Bianchi P G, Manicardi G C, Bizzaro D, Bianchi U and Sakkas D 1993 Biol. Reprod. 49 10838 [35] Martin S R and Schilstra M J 2008 Methods Cell Biol. 84 26393 [36] Johnson W C Jr 1996 Determination of the conformation of nucleic acids by electronic CD Circular Dichroism and the

[37] [38] [39] [40]

Conformational Analysis of Biomolecules (New York: Plenum) Kimoto H and Taketo A 1996 Biochim. Biophys. Acta 17 32530 Barry M E, Pinto-Gonz alez D, Orson F M, McKenzie G J, Petry G R and Barry M A 1999 Hum. Gene Ther. 10 246180 Conwell C C and Huang L 2005 Adv. Genet. 53PA 118 Je J Y, Cho Y S and Kim S K 2006 Biomacromolecules 7 344851