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Bioconjugate Chem. 2010, 21, 836–843
Low Molecular Weight Polyethylenimine Conjugated Gold Nanoparticles as Efficient Gene Vectors Chu Hu, Qi Peng, Fujie Chen, Zhenlin Zhong,* and Renxi Zhuo* Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China. Received August 22, 2009; Revised Manuscript Received March 7, 2010
Gold nanoparticles (GNPs) conjugated with low molecular weight polyethylenimine (PEI 800 Da) were synthesized, and their characteristics as gene transfection vectors were investigated. The polyethylenimine conjugated GNPs (GNP-PEI800s) can retard plasmid DNA completely at N/P ratios above 4 in electrophoresis on agarose gel, and they also render effective protection of DNA from attack by DNase. TEM imaging revealed that GNP-PEI800s with higher PEI grafting density resulted in more compact and smaller complexes with plasmid DNA, compared to those obtained with lower grafting density ones. These complexes showed high efficiency in gene delivery in monkey kidney cells in vitro. In the absence of serum, GNP-PEI800s can transfect pGL-3 to COS-7 cells 3 to 4 orders more efficient than unmodified PEI800, reaching almost the same magnitude of PEI 25 kDa. More importantly, in contrast to the dramatically lowered efficiency of high molecular weight PEIs such as PEI 25 kDa in the presence of serum, the efficiency of GNP-PEI800s can be retained or even enhanced in serum-containing media. GNP-PEI800 1.3 exhibited transfection efficiency exceeding 60-fold that of PEI 25 kDa in 10% serum medium. All GNP-PEI800s exhibit mild cytotoxicity in comparison with that of PEI 25 kDa.
INTRODUCTION Substantial effort has been focused on synthetic nonviral vector systems such as cationic lipids (1), dendrimers (2), and cationic polymers (3). Among them, polyethylenimine (PEI) is one of the most extensively studied polycations (4). The relatively high transfection efficiency of PEI has led to intense research in terms of both understanding (5, 6) and improving (7, 8) gene delivery by PEI. The efficiency and toxicity of PEI are strongly correlated with its molecular weight (MW) (9). High molecular weight PEI such as PEI with molecular weight of 25 000 Da (PEI 25 kDa) leads to high transgene expression, but its acute cytotoxicity and significantly lower efficiency in the presence of serum (10) has restricted its clinical development (11). Meanwhile, low molecular weight PEI, which has been demonstrated to be nontoxic in cell culture studies, displays very poor transfection activity (12), presumably because of its inability to condense DNA effectively (13). To achieve a useful level of transfection efficiency while minimizing the toxicity, low molecular weight PEIs such as PEI with molecular weight of 800 Da (PEI800) are modified by cross-linking via biodegradable linkages (14-16). Recently, efforts have been made for construction of inorganic nanoparticles as alternate nonviral vectors including silica (17), iron oxide (18), carbon nanotube (19), and gold nanoparticles (GNPs) (20-24). GNPs provide attractive scaffolds for gene delivery vectors (25-29). GNPs can be readily synthesized varying in diameter from about 5 nm to around 150 nm and conjugated with targeting molecules through thiol linkages. GNPs of small size provide a high surface-to-volume ratio, maximizing grafting density of target molecules, which allow further tuning of the surface charge and hydrophobicity. Moreover, GNPs are bioinert and nontoxic (30). Various GNPs functionalized with cationic quaternary ammonium (20), cationic lipid (21), chitosans (22), amino acids (23), and polyethylen* To whom correspondence and reprint requests should be addressed. Phone: +86-27-6875-4061. Fax: +86-27-6875-4509. E-mail: zlzhong@ whu.edu.cn.
imine (24) were demonstrated to be excellent vehicles for the delivery of genetic material into cells. Thomas and Klibanov covalently conjugated branched PEI of 2 kDa to GNPs and found that conjugation to GNPs greatly enhances PEI’s transfer of plasmid DNA into mammalian cells (24). We recently reported a convenient procedure for preparation of thiolated PEI with controllable amounts of thiol groups by ring-opening reaction of thiiranes (16a) and found that disulfide cross-linked PEI800 is superior to the cross-linked PEI 1.8 kDa and 25 kDa for use as gene vectors (16b). Efficient gene delivery systems based on low molecular weight PEIs such as PEI800 are attractive because of lower toxicity and easier clearance from body than higher molecular weight ones. In the present study, we devised a series of PEI800 conjugated GNPs (GNP-PEI800) at different PEI/Au molar ratios as nonviral vectors. The effect of PEI grafting density on DNA binding capacity, DNA protection ability, surface charge and size of GNP-PEI800s/DNA complexes, cytotoxicity, and in vitro transfection efficiency of nanoparticles were investigated.
MATERIALS AND METHODS Materials. Branched PEI with molecular weights of 25 kDa and 800 Da were purchased from Aldrich. Methylthiirane was synthesized by literature procedure (31). HAuCl4 tetrahydrate and NaBH4 were obtained from Shanghai Chemical Co (China). GelRed was purchased from Biotium (CA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), Dulbecco’s phosphate buffered saline (PBS), and 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Invitrogen Corp. The Micro BCA protein assay kit was purchased from Pierce. DNase, DNase reaction buffer, and stop reaction buffer were purchased from (Promega, USA). Ultrapure water (18.0 MΩ, Millipore) was used throughout the experiment. Plasmid pGL3 was propagated at 37 °C in Escherichia colia in Luria-Bertani (LB) medium containing 50 µg/mL kanamycin or 60 µg/mL ampicillin. The mixture was shaken overnight at 250 rpm. The plasmid was purified using E.Z.N.A. Fastfilter Endo-Free Plasmid Midi Kits (Omega)
10.1021/bc900374d 2010 American Chemical Society Published on Web 05/03/2010
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Table 1. Composition and Characteristics of PEI Conjugated Gold Nanoparticles GNP-PEI800 composition sample
molar feed ratioa (Au:PEI)
PEIb(wt%)
Auc(wt%)
molar ratio (Au:PEI)
gold core sized (nm)
number of PEI800 molecules per particle
number of gold atoms per particle
grafting density (NPEI/nm2)
GNP-PEI800 5.6 GNP-PEI800 4.3 GNP-PEI800 1.3
1:1.6 1:3.1 1:15
21.7 25.2 35.6
28.4 25.4 10.3
5.6:1 4.3:1 1.3:1
5.7 5.4 7.7
1000 1100 11000
5600 4900 14000
10 12 57
a Molar feed ratio of HAuCl4 · 4H2O to PEI 800 Da. measurement. d Based on TEM images.
b
Calculated on the basis of nitrogen content from elemental analysis.
according to the manufacturer’s instruction. The DNA concentration was determined by measuring the UV absorbance at 260 nm. The purity of DNA was assessed spectrophotometrically by measuring absorbance at wavelengths of 260 and 280 nm (OD260/OD280 1.8 or greater). DNA aliquots were stored at -20 °C prior to use. Synthesis of Thiolated PEI (PEI-SH). In a 25 mL flask, 1.00 g of PEI800 was dissolved in 5 mL of deionized water. Hydrochloric acid (0.1 M) was added to the PEI solution until pH was regulated to 7.2. After removal of water, the neutralized PEI was redissolved in 10 mL of CH3OH. The assigned amount of thiirane was added after the container was purged with argon. The flask was kept in a 50 °C oil bath for 24 h under magnetic stirring. The solution was evaporated to remove CH3OH and the yellow solid thus obtained was kept under argon. Two kinds of thiolated PEIs varying in thiol group content were synthesized, and the thiol group contents were determined to be 0.8 and 1.7, respectively, by Ellman’s method (32, 33). General Procedure for the Synthesis of GNP-PEI800. HAuCl4 (tetrahydrate, 0.10 g, 0.24 mmol) was mixed with calculated amount of PEI-SH in 10 mL of water and stirred for 15 min; then, freshly prepared NaBH4 solution (0.45 g, 12 mmol in 5 mL of water) was added dropwise. Within 90 s, the solution turned dark purple signaling the onset of gold nanoparticles formation. The mixture was stirred at room temperature for an extra 24 h. The conjugates were dialyzed extensively against water with 12 kDa cutoff dialysis tubes. The solution was filtered through a 0.22 mm pore size polyether sulfone membrane and stored at room temperature for use. A measured portion was lyophilized and submitted for elemental analysis. On the basis of nitrogen content from elemental analysis and gold content from ICP measurements, the ratios of gold/PEI800 were calculated and summarized in Table 1. Instrumentation. Elemental analysis was performed by using Vario EL III (Germany). UV-vis spectra were recorded on a Perkin Elmer UV-vis spectrophotometer with a quartz cuvette with a 10 mm optical path length. The ICP-AES was carried on a Thermo IRIS Intrepid II XSP spectrometer (USA). The morphologies of GNP-PEI800s and GNP-PEI800/DNA complexes were observed on transmission electron microscope (TEM JEM-100CXa) with an acceleration voltage of 100 kV. Particle Size and Zeta Potential Measurements. The particle size and zeta potential were measured with a Nano-ZS ZEN3600 (Malvern) at room temperature. The relative amount of GNP-PEI800s/DNA was express as N/P molar ratio which is calculated with the N content in GNP-PEI800s based on the elemental analysis and the P content in the DNA solution of known concentration. The GNP-PEI800s/DNA complexes at various N/P ratios ranging from 30 to 180 were prepared by adding appropriate volumes of GNP-PEI800s to 50 µL of pGL-3 DNA (20 ng/mL in 40 mM Tris-HCl buffer solution), and then the complexes were diluted to a total volume of 1 mL with 150 mM NaCl and vortexed for 15 s. After that, the complexes were incubated at room temperature for 30 min before measurements of particle sizes or zeta potentials. Agarose Gel Retardation Assay. GNP-PEI800/DNA complexes at predetermined N/P ratios were loaded on 0.7% (w/v)
c
Based on ICP-AES
agarose gel containing GelRed and electrophoresed with Trisacetate (TAE) running buffer at 80 V for 80 min. DNA was visualized with a UV lamp using a Vilber Lourmat imaging system (France). DNase Protection Assay. The concentration of DNase enzyme was 1 U/µg DNA. Reaction mixture for DNase digestion contained 1.0 µL of 10× reaction buffers, 8.0 µL of either DNA or GNPPEI800s/DNA complex with an amount of DNA of 0.2 µg, and 1.0 µL of DNase enzyme (1 U/µL). The digestions were incubated at 37 °C for 30 min and halted by addition of 1.0 µL of stop solution followed by heating at 65 °C for 30 min. The complexes were then analyzed by agarose gel electrophoresis. The integrity of DNA in each formulation was compared with untreated DNA as a control. Cytotoxicity Assay. The cytotoxicity assay was carried out on the basis of an MTT assay on COS-7 cells. Cells were seeded in 96-well plates at an initial density of 5000 cells/well in DMEM complete medium. The cells were allowed to grow for 24 h. The original media were then replaced with 100 µL of fresh DMEM complete media. The GNP-PEI800s, PEI 800 Da, or PEI 25 kDa solution were added to the media. Each dosage was replicated in 4 wells. Treated cells were incubated at 37 °C under a humidified atmosphere of 95% air and 5% CO2 for 24 h. MTT reagent (20 µL in PBS, 5 mg/mL) was added to each well, and the cells were incubated for another 4 h at 37 °C. The liquid in each well was removed with cautions to avoid disturbing the crystals on the well wall. 100 µL of DMSO was added to each well to dissolve the crystals. The absorbance at 570 nm of the solution in each well was recorded using a Microplate Reader (Bio-Rad model 550). In addition to the general control experiment of cell culturing in the absence of the polycation samples, control experiment using GNP-PEI800s in medium without cells was carried out to exclude the interference caused by the absorbance of gold nanoparticles at 570 nm. Cell viability was calculated according to the following equation: Cell viability (%) ) (ODsample ODGNP - ODblank)/(ODcontrol - ODblank) × 100 where ODsample is the absorbance of the solution of the cells cultured with GNPPEIs and PEI; ODblank is the absorbance of the medium, ODcontrol is the absorbance of the solution of the cells cultured with the medium only, and ODGNP is the absorbance of the control solution of GNP-PEI800s in medium without cells. Luciferase Assay. For in vitro transfection study, COS-7 cells were plated in 24-well plates at a density of 5 × 104 cells/well. They were incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO2 until the cells reached about 70% confluence. At the time of transfection, the medium in each well was replaced with 1 mL of DMEM without FBS. The cells were transfected with gene-loaded complexes containing 1 µg of plasmid DNA at 37 °C for 4 h. Then, the complexes were removed, and the cells were washed by PBS three times. The cells were then incubated in fresh DMEM with 10% FBS at 37 °C for another 48 h and lysed with the reporter lysis buffer (Promega, USA). The luciferase activity in cell extracts was measured using a luciferase assay kit (Promega, USA). The relative light units (RLU) were normalized by the total protein concentration of the cell extracts, and the total protein was
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Scheme 1
measured with a BCA protein assay kit (PERBIO, USA). Luciferase activity was expressed as RLU/mg protein.
for an extra 4 h, the cells were directly visualized on a laser confocal scanning microscope (Eclipse C1Si, Nikon, Japan).
Cell Uptake Tracing Assay. GelRed was used as molecular probe to label pGL-3. The GNP-PEI800s/DNA complexes at N/P ratio of 90 were prepared. After the complexes were incubated for 4 h at 37 °C, the medium was replaced with GelRed solution (in 1/10 000 (v/v) DMEM with 10% FBS) and incubated for 10 min at 37 °C. Then, the GelRed solution was replaced with fresh DMEM with 10% FBS. After incubating
RESULTS AND DISCUSSION
Figure 1. Absorption spectra of (a) GNP-PEI800 5.6, (b) GNP-PEI800 4.3, and (c) GNP-PEI800 1.3 at concentration of 0.15 mg/mL.
Preparation and Characterization of GNP-PEI800s. As shown in Scheme 1, thiolated PEIs (PEI-SH) were synthesized via the ring-opening reaction of methylthiirane on low molecular weight PEI with an Mw of 800 Da. PEI conjugated golden nanoparticles (GNP-PEI800s) were prepared by the simple onepot reduction of tetrachloroaurate with sodium borohydrate in the presence of PEI-SH. To explore the effect of PEI grafting density on transfection efficiency, we fabricated GNPs with varied Au/PEI molar feed ratios. The elemental composition of the GNP-PEI800s was measured by using a Vario EL III elemental analyzer (for CHN) and ICP-AES measurement (for Au). On the basis of the nitrogen and gold contents, the molar ratio composition of the GNP-PEI800s was calculated and summarized in Table 1. The UV-vis spectra of GNP-PEI800s were shown in Figure 1. These spectra have a strong absorption feature at 520 nm due to the characteristic surface plasmon resonance of GNPs (34). Though the GNP-PEI800s were at the same weight concentration of 0.15 mg/mL, the absorbance strength was different, because the absorbance of the conjugates is proportional to the content of GNP cores other than the weight of the total conjugates. Actually, the absorbance strength in Figure 1 is in good agreement with the Au/PEI composition ratios in Table 1. Electron micrographs of GNP-PEI800s and those of their complexes with the plasmid pGL3 DNA are presented in Figure 2. All PEI800-GNPs exhibited no apparent
Figure 2. TEM micrographs of (A) GNP-PEI800 5.6, (B) GNP-PEI800 4.3, (C) GNP-PEI800 1.3, (D) GNP-PEI800 5.6/pGL3 complex, (E) GNPPEI800 4.3/pGL3 complex, and (F) GNP-PEI800 1.3/pGL3 complex. The GNP-PEI800/pGL3 complexes were prepared at N/P ratio of 30.
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signs of aggregation. The sizes of the gold core deduced from TEM were 5.7 ( 1.5 nm for GNP-PEI800 5.6, 5.4 ( 0.8 nm for GNP-PEI800 4.3, and 7.7 ( 1.8 nm for GNP-PEI800 1.3, respectively. GNP conjugates with thiolated PEI 2 kDa were previously prepared and studied for enhanced gene tansfection (24). Compared with the literature procedure of making thiolated PEI by reduction of disulfide cross-linked PEI, our thiirane ringopening procedure is more direct and controllable without need of time-consuming dialysis. The GNP-PEI800s have higher PEI content that is beneficial to higher gene loading capacity. It is worth noting that GNP-PEI800s are very stable. After 6 months of stocking at room temperature, no obvious changes were observed with GNP-PEI800 solutions. Determination of Graft Density of PEI800 on GNPs. The number of gold atoms in each nanoparticle (U) was calculated according to eq 1 (35).The number of PEI molecules per nanoparticle (N) and grafting density of PEI (δ) can be determined using eqs 2-5, and the results are shown in Table 1. U)
2 D π 3 R
(1)
WAu MAuU
(2)
WPEI MWPEI
(3)
NGNPs )
NPEI )
3
()
Figure 3. Particle size of GNP-PEI800/pGL3 complexes at various N/P ratios in 150 mM NaCl solution.
NPEI NGNPs
(4)
N πD2
(5)
Figure 4. Surface charge of GNP-PEI800/pGL3 complexes at various N/P ratios with fixed pGL3 concentration of 1 µg/mL.
where R refers to the edge of a unit cell, which has a value of 4.0786 Å on the edge; there are four gold atoms per unit cell. D is the diameter of nanoparticles. WAu and WPEI are the gold content and PEI content, and NGNPs and NPEI are the molar number of gold nanoparticles and PEI. MWPEI and MAu are the molecular weight of PEI and gold. Characterization of Complexes. The morphology of the complexes was also monitored by TEM. Upon the addition of DNA, GNP-PEI conjugates tended to aggregate with DNA forming submicrometer particles. At N/P ratio of 30, all of the complexes exhibited diameters below 400 nm. The structure of the complexes varied as a function of PEI grafting density on the GNPs. While mainly anomalous loose aggregations with a size of around 400 nm were observed in the images of GNPPEI800 5.6/DNA (Figure 2D), both anomalous loose and spherical structures were found in GNP-PEI800 4.3/DNA (Figure 2E), and when GNP-PEI800 1.3/DNA complexes were formed, compact and ordered spherical granules were the primary structure as shown in Figure 2F. The particle sizes of the complexes were further determined by DLS (Figure 3). At low N/P ratios, the complex size decreases rapidly with the increase in the amount of GNPPEI800. At higher N/P ratios, however, the size becomes less sensitive or even independent of the ratio. As for GNP-PEI800 4.3 and GNP-PEI800 1.3, the sizes were around 200 nm and fairly consistent at N/P ratios ranging from 30 to 180. For complexes with GNP-PEI800 5.6, the particle size showed no obvious differences at high N/P ratios over 90. This phenomenon could be interpreted as the complexation of DNA by the GNPPEI800 conjugate is saturated above a certain N/P ratio. The results of size measurement by DLS were in accordance with
TEM results that a higher PEI grafting density would promote condensation more efficiently. Surface charge of DNA complexes is another important parameter in determining the efficiency of cellular uptake. Zeta potentials of the complexes were shown in Figure 4. Regardless of the Au/PEI composition, all DNA complexes of the three GNP-PEI800 conjugates showed very similar surface charge profiles. At very low N/P ratio of 1, the particles had zeta potential of about -10 mV, implying that the negative charge of DNA is not completely neutralized. With increasing N/P ratio, zeta potentials of the complexes increased and reached plateaus between +30 and +40 mV at N/P ratios above 5. Complexes of GNP-PEI800s bearing positive surface potentials that should promote initial adhesion on a negatively charged cell surface (31). The ability of GNP-PEI800s to interact with DNA was characterized by agarose gel electrophoresis. In good agreement with zeta potential measurements, the migration of pDNA was completely retarded at a relatively low N/P ratio of 4 (Figure 5), which indicated high DNA binding capacity of all the conjugates. Intensities of DNA staining decreased obviously at higher N/P ratios. Condensation of DNA with GNP-PEI800s might result in exclusion of the dye due to alterations in the double helix leading to reduced fluorescence emission (37). As a result, the intensities of DNA staining decrease with increasing DNA condensation ability at higher N/P ratios. To investigate whether GNP-PEI800s are efficient in protecting DNA from enzyme cleavage, an in vitro DNase protection assay was performed by agarose gel electrophoresis. An amount of enzyme equal to 5 units (1 U/µg DNA) was fixed in the experiment. DNase was added to naked plasmid DNA and GNPPEI800/DNA complexes (N/P ) 20), followed by incubation
N)
δ)
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Figure 7. Cell viability at 24 h after the addition of GNPs, PEI 25 kDa, and PEI 800 Da.
Figure 5. Agarose gel electrophoresis assay of the complexes of pGL3 plasmid DNA (100 ng/well) with (A) GNP-PEI800 5.6, (B) GNPPEI800 4.3, and (C) GNP-PEI800 1.3 at various N/P ratios. The line of N/P ) 0 refers to pGL3 in the absence of GNP-PEI800.
Figure 8. In vitro transfection efficiency of complexes of pGL3 with GNPs in COS-7 cells in comparison with PEI 25 kDa and PEI 800 Da in serum-free medium.
Figure 6. Agarose gel showing DNase digestion results. Lanes 1, 3, 5, 7 are undigested samples of naked DNA, GNP-PEI800 5.6/DNA, GNPPEI800 4.3/DNA, and GNP-PEI800 1.3/DNA, respectively. Lanes 2, 4, 6, 8 are the above samples exposed to enzyme. Five units of enzyme were used for each reaction. The amount of DNA was 200 ng/well.
for 15 min at 37 °C. As shown in Figure 6 (lane 2), when exposed to the same amount of enzyme under the same conditions, free plasmid DNA alone was digested thoroughly, while plasmid DNA complexed with GNP-PEI800s remained intact. Cytotoxicity. The cytotoxicity of GNP-PEI800s was evaluated by MTT assay. It should be noted that the absorbance of GNP obviously affects the assay accuracy especially at high concentration. Control experiment using GNP-PEI800s in medium without cells was carried out to get rid of the interference of the absorbance of gold nanoparticles on the color intensity of the assay at 570 nm. The results are presented in Figure 7. In the case of GNP-PEI800 5.6 and GNP-PEI800 4.3, half of the cells were still metabolically active when exposed to a concentration of 300 mg/L, which was almost the same as unmodified PEI 800 Da. In contrast, GNP-PEI800 1.3 with higher PEI content showed greatly increased cytotoxicity compared to the other two GNP-PEI800s, though its half-cell viability concentration was still three times higher than that of PEI 25 kDa. The relatively higher cytotoxicity of GNP-PEI 1.3 may be caused by its high PEI grafting density of 57 chains/
Figure 9. In vitro transfection efficiency of complexes of pGL3 with GNPs in COS-7 cells in comparison with PEI 25 kDa and PEI 800 Da in medium containing 10% serum.
nm2. High cationic charge densities and compact structure, as well as high molecular weight, usually result in high cytotoxicity. GNP-PEI800 Conjugates Mediated in Vitro Transfection. Transfection efficiency of GNP-PEI800 conjugates was evaluated by luciferase expression of pGL3 plasmid DNA in monkey kidney (COS-7) cells. Complexes were prepared with N/P ratios ranging from 30 to 180, among which the optimal ratio of each complex was determined. Branched PEI 25 kDa at its optimal N/P ratio of 10 and unmodified PEI 800 Da were used for comparison. Figure 8 shows luciferase expression mediated by the conjugates in the serum-free medium. Under this condition,
GNP-PEI800s as Gene Vectors
Figure 10. In vitro transfection efficiency of complexes of pGL3 with GNP-PEI800 1.3 at different serum concentrations (v/v) in comparison with PEI 25 kDa.
GNP-PEI800 4.3/DNA complexes at N/P ratio of 90 exhibited the highest efficiency, while GNP-PEI800 5.6 and GNP-PEI800 1.3 reached their maxima at N/P ratio of 150 and 60,
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respectively. The optimal N/P ratio of each GNP-PEI800 decreased with the increase of PEI grafting density on the GNPs, indicating that GNPs with higher PEI density may achieve efficient transfection at lower N/P ratios. At its best ratio, GNPPEI800 4.3 achieved nearly half the luciferase expression of PEI 25 kDa, which was worth mentioning, since unmodified PEI 800 was 1000 times less efficient even at a relatively high N/P ratio of 150. In the case of GNP-PEI800 1.3, complexes prepared at N/P ratio of 90 or higher ratios led to abnormal sharply reduced efficiency, possibly ascribed to the increased cytotoxicity, which may also explain why its efficiency was not the highest among the three. It is known that transfection efficiency of high molecular weight PEI decreased in serum supplemented medium (31). Therefore, the development of nonviral gene vectors that can retain efficiency in serum is important. Thus, we tested transfection efficiency of GNP-PEI800s in the presence of serum. As shown in Figure 9, when 10% serum was supplemented in the media, the strong dependence of transfection activity on PEI grafting density became more obvious. The transfection efficiencies declined in the order GNP-PEI800
Figure 11. Confocal cell images showing tracking of GelRed labeled GNP-PEI800/DNA complexes. (A-C) Complexes of GNP-PEI800 5.6/DNA, GNP-PEI800 4.3/DNA, and GNP-PEI800 1.3/DNA at N/P ratio of 90 after 4 h post-transfection in serum-free medium. (G-I) Complexes of GNP-PEI800 5.6/DNA, GNP-PEI800 4.3/DNA, and GNP-PEI800 1.3/DNA at N/P ratio of 90 after 4 h post-transfection in medium containing 10% serum. The light inverted micrographs are D-F and J-L, respectively. The micrographs were obtained at magnification of 400×.
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1.3 > GNP-PEI800 4.3 > GNP-PEI 800 5.6 (at optimal N/P ratios of 60, 90, and 150, respectively). This sequence is consistent with the PEI grafting density of the GNP-PEI800 conjugates. Specifically, GNP-PEI800 1.3 exhibited efficiency 10 times higher than that of GNP-PEI800 4.3 and PEI800-GNP 5.6 at their optimal N/P ratios. Moreover, the efficiency of GNPPEI800 1.3 was found to significantly exceed that of PEI 25 kDa. It is interesting that the presence of serum resulted in significant enhancement of gene transfer efficiency of GNPPEI800 1.3, while GNP-PEI800 4.3 and PEI800-GNP 5.6 could also retain their efficiency under the same conditions. In contrast, the efficiency of PEI 25 kDa was greatly decreased by the presence of serum. To get further insight into the effect of serum on efficiency, we performed transfection at different serum concentrations. Since GNP-PEI800 1.3 exhibited the most striking distinction between its efficiency in the absence and presence of serum, it was selected for the subsequent experiment. Two N/P ratios of 30 and 60 were chosen to exclude the impact of cytotoxicity at higher ratios. Branched PEI 25 kDa at the optimal N/P ratio of 10 was used for comparison. Figure 10 shows the comparative gene transfection efficiencies of GNP-PEI800 1.3 and PEI 25 kDa in the presence of various percentages of serum. There was a gradual decrease in the transfection efficacy with increase in serum concentrations in the case of PEI 25 kDa, while GNPPEI800 1.3 showed a completely opposite tendency. For example, at 6% serum concentration, only 5% of its transfection efficiency in serum-free medium was observed for PEI 25 kDa, whereas gene expression with GNP-PEI800 1.3 was promoted about 7 times higher than that without serum. The result clearly shows that GNP-PEI800 1.3 is a better transfection agent in the presence of serum than PEI 25 kDa. The enhancement of transfection efficiency of GNP-PEI800 1.3 in serum might be related to lowered cytotoxicity. The high PEI grafting density in GNP-PEI800 1.3 can largely increase the effective molecular weight of PEI 800 Da, which consequently leads to more efficient DNA condensation and thus more efficient transfection. However, it induced inevitable cytotoxicity simultaneously. At the same concentration as in its complex, the cell viability of GNP-PEI800 1.3 alone decreased with an increase in N/P ratio, which was greater than 80% cell viability at N/P ratios of 30 and 60, nearly 60% at N/P ratios of 90 and 120, and 20% at N/P ratios of 150 and 180. These results revealed that the cytotoxicity of the GNP-PEI800 conjugates is remarkable under the transfection conditions. Thus, the enhancement of transfection efficiency in serum-containing medium might be related to the cell function raised by the addition of serum. Cell Uptake Tracing. The capacity of GNP-PEI800s to internalize plasmid DNA into cells was additionally observed by confocal laser scanning microscopy (CLSM). GelRed was used as a molecular probe to label pGL-3. Complexes of all three GNP-PEI800 samples at N/P ratio of 90 were incubated in the medium in the absence and presence of serum, respectively, and examined at 4 h post-transfection. Control experiments with labeled naked DNA showed no signs of cellular uptake (data not shown). As shown in Figure 11, the uptake of complexes in the absence and presence of serum exhibited subtle differences. In the case of GNP-PEI800 1.3, the percentage of cells showing complex uptake was high, which probably contributed to its high transfection efficiency. However, the high absorption of GNP-PEI800 1.3 may also impair plasma membrane dramatically and result in high cytotoxicity. In fact, a significant level of cellular dysfunction or even cell death was observed in the images. By comparison of the two images in Figure 11F and L, it is obvious the cells in the serum-containing medium were better in morphology than those in serum-free
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medium. The results imply that the cytotoxicity of the PEI conjugated GNP was reduced by the presence of serum, while the cell uptake capacity was retained. Generally, the qualitative results of CLSM are in good agreement with the results of luciferase assay and cytotoxicity assay.
CONCLUSION In closing, we have synthesized a series of hybrid GNPPEI800s by conjugating PEI 800 Da to gold nanoparticles at three PEI/Au molar ratios. GNP-PEI800s possesses many outstanding features in favor of polycation-mediated gene delivery, including efficient DNA condensation, high DNA binding capability and DNA protection against degradation by DNase. In consequence, GNP-PEI800s can transfect COS-7 cells 3 to 4 orders more efficient than unmodified PEI800, reaching almost the same magnitude of PEI 25 kDa. Moreover, in contrast to the dramatically lowered efficiency of high molecular weight PEIs such as PEI 25 kDa in the presence of serum, the efficiency of GNP-PEI800s can be retained or even enhanced, reaching 60-fold higher than that of PEI 25 kDa in 10% serum medium. The research demonstrated that GNP-PEI conjugates are very promising as gene delivery vectors.
ACKNOWLEDGMENT This research was financially supported by National Natural Science Foundation of China (20774067) and National Basic Research Program of China (2005CB623903 and 2009CB930300).
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