Cationic

Article pubs.acs.org/Langmuir

Enhanced Gene Expression Promoted by Hybrid Magnetic/Cationic Block Copolymer Micelles E. Haladjova,*,† S. Rangelov,† Ch. B. Tsvetanov,† V. Posheva,‡ E. Peycheva,‡ V. Maximova,‡ D. Momekova,§ G. Mountrichas,∥ S. Pispas,∥ and A. Bakandritsos⊥ †

Institute of Polymers and ‡Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria Faculty of Pharmacy, Medical University of Sofia, Sofia 1000, Bulgaria ∥ Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, 48 Vass. Constantinou Ave., 116 35 Athens, Greece ⊥ Department of Materials Science, University of Patras, 265004 Rion, Greece §

ABSTRACT: We report on novel gene delivery vector systems based on hybrid polymer-magnetic micelles. The hybrid micelles were prepared by codissolution of hydrophobically surface modified iron oxide and amphiphilic polystyrene-b-poly(quaternized 2-vinylpyridine) block copolymer (PS-b-P2QVP) in organic solvent. After extensive dialysis against water, micelles with positively charged hydrophilic corona of PQVP and hydrophobic PS core were prepared, in which magnetic nanoparticles were randomly distributed. The hybrid micelles were used to form complexes with linear (salmon sperm, 2000 bp, corresponding to Mw of 1.32 × 106 Da) and plasmid (pEGFP-N1, 4730 bp, corresponding to Mw of 3.12 × 106 Da) DNA. The resulting magnetopolyplexes of phosphate:amine (P/N) ratios in the 0.05−20 range were characterized by light scattering, ζ-potential measurements, and transmission electron microscopy as well as cytotoxicity and gel retardation assays. The investigated systems displayed a narrow size distribution, particle dimensions below 360 nm, whereas their ζ-potential values varied from positive to negative depending of the P/N ratio. The resulting vector nanosystems exhibited low toxicity. They were able to introduce pEGFP-N1 molecules into the cells. The application of a magnetic field markedly boosted the transgene expression efficiency of the magnetopolyplexes, which was even superior to those of commercial transfectants such as Lipofectamine and dendritic polyethylenimine.

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INTRODUCTION Gene therapy refers to treatment of deseases by modifying gene expression within specific cells. It has emerged as a promising approach for treatment of not only genetic diseases but also viral infections and cancer.1−4 The concept behind is very simpleexogeneous delivery of therapeutic genes to the body, the most significant issue and still a great challenge for which is the development of an efficient delivery system. The fundamental criteria for any gene delivery system require the ability to (i) package therapeutic genes, (ii) gain entry into cells, (iii) escape the endolysosomal pathway, (iv) effect gene/ vector release, (v) traffic through the cytoplasm and into the nucleus, and (vi) enable gene expression.5 The vector systems are generally divided into viral and nonviral. Engineered viruses were the first gene delivery systems.6 The viral vectors are characterized by very good efficiency; however, they have shown very high toxicity7,8 and appeared quite risky to the patient9−12 as severe side effects have been made strikingly evident.13 The nonviral vectors have recently attracted much attention since they are less hazardous, considerably safer, less pathogenic, and less immunogenic compared to the viral ones.5,14,15 Various synthetic systems including inorganic particles,16,17 cationic lipids,18 and liposomes19 as well as © 2014 American Chemical Society

polymers and polymer-based particles have been used as nonviral carriers. The systematic delivery to various tissues, including tumors, demands the carrier to stabilize and transport the genetic material to the target cells. In particular, the ability (i) to neutralize the negatively charged DNA molecules in order to prevent repulsion from the anionic cell surface, (ii) to condense the bulky structure of DNA chains to suitable size in order to pass through the cell wall, and (iii) to protect the DNA chains from degradation in the cell are considered of primary importance for any synthetic vector system.5,20 Most of the polymeric vectors (linear, branched, and dendritic polymers21−23 as well as copolymers) developed so far act by complexation via electrostatic interactions between the negatively charged DNA and positively charged moieties of the polymer to neutralize and condense DNA. Copolymer micelles obtained by self-assembly of amphiphilic block copolymers have also been reported to shrink DNA macromolecules.24−26 The unique core−shell structure of the micellesa hydrophobic nonionic core and a hydrophilic Received: December 13, 2013 Revised: June 10, 2014 Published: June 19, 2014 8193

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also used. The cultivation medium was minimum essential medium (MEM) EAGLE and 10% fetal bovine seruum from BIOCHROM. Methods. Preparation of Magnetic Nanoparticles. The iron precursor was prepared by dissolving 2 g of FeCl3 in 20 mL of water. Then 19 mL of 1 M NaOH was added, and the solution was centrifuged at 4000 rpm for 5 min. The precipitated iron hydroxide was redispersed in H2O and washed until complete removal of Cl ions (AgNO3 test). Washing continued by sequential dispersion of precipitants in ethanol and acetone in order to remove water. The obtained iron hydroxide (1.2 g), oleic acid (4.31 g), and benzyl ether (30 mL) were introduced in a flask. The mixture was kept at 270 °C for 30 min. After that, acetone at a 1:1 volume ratio was added and the product was collected with a magnet. According to thermogravimetric analysis, the product contained 65 wt % Fe2O3. The mean size of magnetic nanoparticles determined by TEM was 4 nm (Figure 1a). Preparation of PS-b-PQ2VP Micelles Loaded with Magnetic Nanoparticles. The amphiphilic polystyrene-b-poly(quaternized 2vinylpyridine) block copolymer (PS-b-PQ2VP) was dissolved in dimethylformamide (DMF) at a concentration of 0.5 mg/mL (1 mL). A dispersion of iron oxide nanoparticles in CHCl3 (60 μL), stabilized with oleic acid, was added to the solution. The concentration of magnetic nanoparticles was 10 wt % with regard to the PS block. 1 mL of the resulting solution was added to 5 mL of distilled water (DMF:H2O v/v 1:5) and then dialyzed using Spectrapore 7 membranes with a MWCO of 3500. After extensive dialysis against water for 7 days, micelles with positively charged hydrophilic shell of PQ2VP and hydrophobic PS core loaded with magnetic nanoparticles were obtained. Preparation of Magnetopolyplexes. The magnetopolyplexes were formed by dropwise addition of aqueous dispersion of the hybrid micelles into DNA aqueous solution (100 μg/mL) at ambient temperature under vigorous stirring. The amount of copolymer and DNA solutions were such to give a phosphate/amine groups ratio in the 0.05−20 range. Dynamic and Electrophoretic Light Scattering. The measurements were carried out at 25 °C on a Zetasizer Nano-ZS instrument (Malvern Instruments), equipped with a He−Ne laser (λ = 633 nm) at a scattering angle of 173°. The ζ-potentials were calculated from the obtained electrophoretic mobility at 25 °C by the Smoluchowski equation:

corona of positively charged chainsmimicks the globular proteins, whereas their polyplexes with DNA can be considered as mimetics of histone/DNA complexes formed under physiological conditions in living cells.27 Gaining entry into the cells and trafficking through the cytoplasm and into the nucleus are largely diffusion-limited processes under standard cell culture conditions. To enhance the efficiency of gene delivery, a new approach for transfection in the presence of a magnetic field, called magnetofection,28−30 has been developed. The approach consists of preparation of magnetic vectors, that is, vector assemblies containing magnetic, usually iron oxide, nanoparticles. Under the action of a magnetic force, they are accumulated and/or held in a target tissue against the hydrodynamic forces. In a cell culture, these vectors are magnetically sedimented on the target cells. Thus, the diffusion barrier to nucleic acid delivery may be overcome. Via magnetofection the efficiency of gene delivery can be enhanced up to several hundred fold.28−31 In this study, we report on preparation of hybrid vector nanosystems, based on cationic block copolymer micelles loaded with magnetic nanoparticles. These magnetic micelles interact via electrostatic interactions with DNA molecules and form nanosized complexes (magnetopolyplexes) that possess magnetic properties, allowing them in the presence of a magnetic field to target and concentrate on the specific location in the body. Complete physicochemical and biopharmaceutical characterization of the hybrid vector nanosystems is accomplished.

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EXPERIMENTAL SECTION

Materials. Salmon sperm DNA (D-1626, 2000 bp, corresponding to Mw of 1.32 × 106 Da) and hyperbranched polyethylenimine (Mw = 25 000 Da) were received from Sigma-Aldrich. The transfection reagent Lipofectamine 2000 was purchased from Invitrogene. Plasmid DNA containing the gene encoding for the enhanced green fluorescent reporter protein pEGFP-N1 (4730 bp, corresponding to Mw of 3.12 × 106 Da) was isolated from glycerin culture E. coli DH5 alpha. For the preparation of the polyplex a stock solution of 100 μg/mL in ultra pure water (>18 MΩ) was used. The polystyrene-b-poly(2-vinylpyridine) block copolymer (PS-b-P2VP-115K) was synthesized by anionic polymerization following the procedure described elsewhere.32 The quaternization of the P2VP block was performed by adding methyl iodide to the copolymer solution in tetrahydrofuran.32 The molecular characteristics of the block copolymer are given in Table 1.

ζ = 4πηυ/ε where η is the solvent viscosity, υ is the electrophoretic mobility, and ε is the dielectric constant of the solvent. Transmission Electron Microscopy. The samples were examined using a HRTEM JEOL JEM-2100 electron microscope operating at 200 kV. They were prepared by depositing a drop of the solution onto a carbon grid. Agarose Gel Electrophoresis. The samples containing linear or plasmid DNA were analyzed by electrophoresis on a 0.7% w/v agarose gel. They were visualized at 260 nm by staining the gel with 0.5 μg/mL ethidium bromide. Cytotoxicity Assay. The toxicity of the prepared magnetopolyplexes was evaluated by two alternative methods: MTT-dye reduction assay and Neutral red uptake (NR) assay. The MTT assay was performed as described previously33 with minor modifications.34 Exponentially growing cells were plated in 96-well sterile plates at a density of 104 cells/well in 100 μL of medium and were incubated for 24 h. Thereafter, the cells were treated with the tested compounds and incubated for 72 h, whereby for each concentration a set of 8 wells was used. After a 72 h continuous exposure period, 10 μL aliquots from a 5 mg/mL MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) solution in sterile phosphate buffered saline were added to each well, and the plates were further incubated for 4 h at 37 °C in a humidified 5% CO2 atmosphere. The formazan crystals yielded were solubilized by addition of HCOOH (5%) acidified isopropanol. The MTT-formazan absorbance was read at 570 nm on a microprocessor controlled multiplate reader (Labexim LMR-1). The cytotoxicity data were processed using GraphPad Prizm software. For the statistical

Table 1. Molecular Characteristics of the PS-b-P2QVP-115K Block Copolymer and Micellar Characteristics of the Micelles Formed in Aqueous Solution (Data Taken from Ref 32) block copolymer

block copolymer micelles

Mw (g mol−1)

Mw/Mn

PS content (wt %)

Nagg

Dh (nm)

ζ (mV)

115000

1.02

44

86

108

26

Cell Lines and Culture Conditions. The cell lines K562 (eritromyeloid leukemia) and MDA-MB-231 (glandular epithelium) were supplied by DSMZ GmbH, Germany. EJ-28 (urinary bladder carcinoma cells) originated from the American Type Cell Culture, USA. Cells were cultured routinely in a controlled environment at 37 °C in a 5% CO2 humidified atmosphere. All cell lines were maintained in RPMI 1640 supplemented with 2 mM L-glutamine and 10% fetal calf serum. All cell lines were subcultured twice weekly to maintain continuous logarithmic growth. EJ-28 were used before the seventh passage. WISH ATCC CCL-25 (human amnion epithelial cells) was 8194

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Figure 1. TEM micrographs of initial iron oxide magnetic nanoparticles (a) and PS-b-PQ2VP-115K micelles loaded with magnetic nanoparticles (b, c). analysis one-way ANOVA was used. Differences were considered significant at the level of p < 0.05. For the Neutral red assay WISH cells were exposed to the different magnetopolyplexes with incorporated linear and cyclic DNA in series of dilutions in a growth medium (MEM with 10% bovine serum). After 24 h incubation at 37 °C at 5% CO2 cells survival was determind by coloring with Neutral red, and the results were quantified using an ELISA READER at 492 nm. Transfection and Magnetofection. The multiply cell line WISH was used which was maintained at 37 °C and 5% CO2 in MEM enriched with 10% bovine serum. Transfection and magnetofection were carried out in six-well plates. In advance, a monolayer of cells (24 h incubation at 37 °C) was prepared on roof glass. Magnetopolyplexes with the plasmid DNA were added to the cells. In the magnetofection experiments, samples (cells and magnotopolyplexes) were put for 5 min in magnetic field and the medium was added. During transfection experiments cells were incubated with magnetopolyplexes for 5 h at 37 °C, and the medium was added. After 24 h incubation at 37 °C the cells were investigated by fluorescent microscope for expression of green fluorescence protein. The intracellular uptake capacity of the studied vectors was investigated by flow cytometry. Samples for fluorescence-activated cell sorting (FACS) survey have been prepared by cells incubation with magnetopolyplexes for 24 h at 37 °C in a minimum quantity of buffered physiological solution. The granularity, size, and fluorescence were determined by BD FACSCalibur TM system.

copolymers have been shown to spontaneously form complexes with DNA in a wide interval of phosphate to amine (P/N) ratios.32,36 The hybrid micelles also spontaneously formed complexes with DNA by electrostatic interactions between the positive ammonium groups in the corona and the negatively charged phosphate groups of DNA. The whole process was initially investigated with linear DNA in order to get basic information about the behavior of the magnetopolyplexes before carrying out biological experiments. Magnetopolyplexes were prepared by dropwise addition of a dispersion containing PS-b-PQ2VP-115K micelles loaded with magnetic nanoparticles to a DNA aqueous solution at room temperature. They were prepared at various P/N ratios in the 0.05−20 range. The size, stability, and surface potential as well as other parameters of the polyplexes are strongly dependent on the P/ N ratio.24−26 The variations of the dimensions and ζ-potential of the resulting magnetopolyplexes are shown in Figure 2.

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RESULTS AND DISCUSSION Preparation of Hybrid Micelles. The self-assembly of the amphiphilic polystyrene-b-poly(quaternized 2-vinylpyridine) (PS-b-P2QVP) block copolymer used in the present study has been thoroughly studied.28,35,36 In aqueous solution the copolymer has been found to form well-defined spherical micelles with a hydrophobic core of polystyrene and a positevely charged corona of poly(quaternized 2-vinylpyridine). The characteristics of the micelles are given in Table 1. In order to impart magnetic properties, these micelles were loaded with small magnetic nanoparticles. The loading was achieved by adding small iron oxide nanoparticles stabilized with oleic acid (Figure 1a) to a copolymer solution in DMF followed by extensive dialysis against water in order to remove the organic solvent. The concentration of magnetic particles was 10 wt % with regard to the PS block. The resulting hybrid micelles were visualized by TEM (Figure 1b,c). The micrographs reveal spherical particles with distinguishable cores and corona in which domains of higher electron density (presumably magnetic nanoparticle) are randomly distributed. Micelles that do not contain magnetic nanoparticles are observable as well. The hybrid micelles exhibited strongly positive values of the ζ-potential (46 mV) and dimensions (an average hydrodynamic diameter) of about 124 nm. Complexation with DNA. Magnetopolyplexes with Linear DNA. The cationic micelles of PS-b-PQ2VP block

Figure 2. Variations of the hydrodynamic diameter (Dh) and ζpotential of magnetopolyplexes formed from PS-b-PQ2VP-115K micelles loaded with magnetic nanoparticles and linear DNA with the P/N ratio.

Precipitation, due to neutralization of the positive and negative charges, occurred in the range of P/N ratios 0.6−0.8. Outside the precipitation region the dimensions varied from about 105 to 350 nm in diameter. The magnetopolyplexes prepared at P/ N ratios close to the precipitation region (0.33−0.6 and 0.8−6) were characterized by small dimensions (typically about 100 nm) and relatively narrow size distributions (PDI in the 0.1− 0.2 range) in contrast to those at both higher and lower P/N ratios (Figure 2). The results obtained from the ζ-potential measurements show that upon increasing the P/N ratio the ζpotential change from positive to negative. The transition is rather sharp and abrupt around the precipitation region. Another stepwise decrease in the ζ-potential was observed at P/ 8195

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physicochemical characterization of the hybrid vector nanosystems are presented in Table 2. The dimensions of the

N > 6, implying the appearance of increasing amounts of uncomplexed DNA. The resulting magnetopolyplexes were visualized by TEM. Figure 3 shows representative micrographs of the magneto-

Table 2. Hydrodynamic Diameters, Polydispersity, and the ζ-Potential of Magnetopolyplexes Formed from PS-bPQ2VP-115K Micelles Loaded with Magnetic Nanoparticles and Plasmid DNA at Different P/N Ratios code

p3

p0.5

p0.33

p0.16

P/N Dh, nm PDI ζ, mV

3/1 149 ± 4 0.33 −33 ± 11

0.5 290 ± 9 0.28 35 ± 7

0.33 302 ± 11 0.25 33 ± 9

0.16 248 ± 6 0.35 29 ± 13

complexes with the plasmid DNA were about 35% larger than those of the corresponding ones prepared with the linear DNA, which could be attributed to the larger size of the plasmid. In addition, the interaction of the plasmid with the magnetic micelles may proceed in a different manner. The plasmid DNA has a cyclic and very rigid structure, which most probably prevents to a large extent the penetration into the micellar corona and interactions with the inner positive charges. The specificities of these interactions can explain the larger PDI values and the less positive ζ-potential. In spite of the different types of interactions, the variations of the ζ-potential, in particular the sharp shift from positive to negative values, follow the general behavior observed also for the complexes with the linear DNA (Table 2). The morphology of the magnetopolyplexes was revealed by TEM. Figure 5 shows spherical objects with dimensions that

Figure 3. TEM micrographs of a magnetopolyplexes obtained from PS-b-PQ2VP-115K micelles loaded with magnetic nanoparticles and linear DNA at P/N = 0.33.

polyplex at P/N = 0.33. It must be noted that the particles retain their spherical shape, and their average diameter is close to that measured by DLS. The TEM images, however, revealed some differences between the structure of the initial magnetic micelles and that of the magnetopolyplexes (cf. Figure 1b,c and Figure 3): whereas the magnetic micelles are characterized by a smooth surface, the surface of the magnetopolyplexes is relatively rough and uneven, which can be attributed to accommodation of the DNA macromolecules on the micellar surface. The magnetopolyplexes were tested using agarose gel electrophoresis (Figure 4). As the negative charges of DNA

Figure 5. TEM micrographs of magnetopolyplexes obtained from PSb-PQ2VP-115K micelles loaded with magnetic nanoparticles and plasmid DNA at P/N = 0.33 (a) and 3 (b).

Figure 4. Agarose gel electrophoresis of magnetopolyplexes formed from PS-b-PQ2VP-115K micelles loaded with magnetic nanoparticles and linear DNA at varying P/N ratios.

are in good agreement with the results from DLS. The rough and uneven structure of the positively charged magnetopolyplexes (Figure 5a) is very similar to that observed for the corresponding complexes prepared with the linear DNA (Figure 2). In the excess of DNA the magnetopolyplexes are composed of several micelles bearing magnetic nanoparticles wrapped with DNA chains (Figure 5b) as previously observed for related systems.32,36 The magnetopolyplexes at P/N ratios from 0.16 to 0.5 did not move through the agarose gel matrix because of their positive ζ-potential (Figure 6). The magnetopolyplex at P/N = 3 and the plasmid DNA marker exhibited bands that ended up at the same distance from the top, which implied that some quantities of uncomplexed DNA were available in that sample. Cytotoxicity. The lack of toxicity is an important prerequisite for the excipients used in preparation of drug delivery systems. Therefore, a basic cytotoxicity screening of investigated systems in a spectrum of four human cell lines was carried out. The lines were selected according to their cell type

were neutralized to varying extents depending on the P/N ratio, corresponding retardation of the magnetopolyplexes upon gel electrophoresis was expected. As seen from Figure 4, the positively charged complexes (that is, those in deficiency of DNA) did not move in the pores of the agarose gel, and characteristic lighting at the starting position was observed. In contrast, the negatively charged complexes were found to migrate through the pores of the gel. Both the linear DNA used as a marker and the complexes at P/N ≥ 3 exhibit indistinguishable smears in their lanes representing multiple unresolved components. Magnetopolyplexes with Plasmid DNA. Experiments were performed with plasmid DNA carrying a specific marker gene pEGFP-N1. Magnetopolyplexes with the micelles loaded with magnetic nanoparticles were prepared in a narrower range of P/ N ratios selected on the basis of the results obtained from the experiments with the linear DNA. The results from the 8196

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structural peculiarities of the copolymer, namely, the presence of a large number of positive charges located within the P2QVP block. The high cytotoxicity of the copolymer, however, is masked upon complexation with DNA as previously shown for other cationic polymers.37 At determining the cytotoxicity by the Neutral red uptake assay similar findings were recorded. The results presented in Figure 8a show that all of the investigated vector systems do not exhibit toxicity at dilutions greater than 1:8. In this case, however, toxicity investigations were carried out on the dispersion of magnetic micelles, rather than on pure copolymer solution. As seen, in contrast to the pure polymer, the hybrid micelles do not show significant toxicity. This may be due to the specific structure, whereby a part of the positive charges are obstructed and screened by the presence of magnetic nanoparticles. The complexes of magnetic micelles and plasmid DNA were also investigated to determine their toxicity. The studies were performed only by the NR assay method in WISH cells, since it was expected that there would be no difference in the results obtained for the vector systems with plasmid DNA. This expectation was confirmed by the results shown in Figure 8b, which demonstrated the absence of toxicity of the tested magnetopolyplexes. Cell Internalization, Transfection, and Magnetofection. Cellular uptake and ability of the hybrid vector systems to transfer pEGFP-N1 into WISH human cells were investigated. The experiments were carried out in the absence and in the presence of an external magnetic field. The results were compared to those obtained at the same experimental conditions with commercial transfection reagents such as Lipofectamine and dendritic polyethylenimine. The internalization was assessed by determination of the size and

Figure 6. Agarose gel electrophoresis of magnetopolyplexes formed from PS-b-PQ2VP-115K micelles loaded with magnetic nanoparticles and plasmid DNA at varying P/N ratios.

as follows: EJ-28 (endothelial cells), K562 (erythro-myeloid cells), MDA-MB-231 (glandular epithelial cells), and WISH (human amnion cells). All models are representative of important cellular populations, which would be exposed to the tested compounds upon systemic administration. The cytotoxicty of different magnetopolyplexes prepared with linear DNA was evaluated by two methods for cell viability (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide [MTT] assays) and Neutral red uptake (NR) assay against nontreated cells. The cytotoxicity of vector systems prepared with plasmid DNA was investigated only by determining the NR assay. The results from MTT-dye reduction assay are presented in Figure 7. Evidently, the tested vector systems did not induce any significant decrease in cell viability with the only exception of the pure copolymer: PS-b-P2QVP-115K exerted strong concentration-dependent cytotoxic effects causing almost total eradication of viable cells at the higher concentration levels. These discrepancies in biological reactivity could be due to the

Figure 7. Cytotoxicity of magnetopolyplexes prepared with linear DNA against EJ-28 (a), K562 (b), and MDA-MB-231 (c) cells, determined by the MTT-dye reduction assay. Each data point represents the arithmetic mean ± SD of eight separate experiments run in triplicate. Co: control cells; P: PS-b-P2QVP copolymer; numbers in the 0.05−20 range correspond to the P/N ratios. 8197

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Figure 8. Cytotoxicity of magnetopolyplexes prepared with linear (a) and plasmid (b) DNA against WISH cells. Each data point represents the arithmetic mean ± SD of eight separate experiments. Co: control cells; MNP: magnetic micelles; numbers correspond to the P/N ratios. The initial concentrations were as follows: Linear DNA: 419 μg/mL (sample 0.05); 368 μg/mL (sample 0.1); 319 μg/mL (sample 0.16); 251 μg/mL (sample 0.33); 215 μg/mL (sample 0.5); 167 μg/mL (sample 1); 125 μg/mL (sample 3); 113 μg/mL (sample 6); 108 μg/mL (sample 10); 104 μg/mL (sample 20). Plasmid DNA: 32 μg/mL (sample p0.16); 252 μg/mL (sample p0.33); 216 μg/mL (sample p0.5); 123 μg/mL (sample p3).

Figure 9. Flow cytometry measurements in the absence (top) and upon application of a megnetic field (bottom) evaluated by size, granularity, and percentage of overall granularity and size of the intracellular uptake of magnetopolyplexes at different P/N ratio: 1, cells; 2, Lipofectamine; 3, PEI (polyethylenimine p0.25); 4, p0.16; 5, p0.33; 6, p0.5; 7, p3.

In general, the fluorescent intensity was low, implying that the WISH cells were not highly susceptible to pEGFP-N1 transfection even if the highly effective Lipofectamine and polyethylenimine were used. In the absence of a magnetic field, the efficiency of the magnetopolyplexes was lower than those of the commercial reagentsless than about 60% as efficient as Lipofectamine. This is in strong contrast with their high internalization level, which is comparable to that of Lipofectamine and higher than that of polyethylenimine (see Figure 9). This finding can be interpreted as indicating that in the absence of a magnetic field the magnetopolyplexes are internalized in the cells; however, they are not able to overcome the next barrier and end up in the endo/lysosomes. Upon the application of a magnetic field, a dramatic increase (up to 3.5 times) in the efficiency of the magnetopolyplexes was observed (Figure 10). The complexes denoted p0.33 and p0.5 exhibited

granularity of the cells. Figure 9 shows the percentage of cells deviating from the normal ones evaluated by size and granularity parameters. As seen, about 45% of the cells changed their size and granularity, which was not different from that of the commercially available Lipofectamine and even better compared to that of polyethylenimine. However, the effect of the magnetic field on the cellular entry was marginal with regard to both commercial reagents and magnetopolyplexes as only slight changes within the standard deviation in the overall size and granularity were detected. This implied that the magnetic forces did not influence the cellular entry. However, once internalized the faith and the effect of the magnetopolyplexes were rather different depending on whether a magnetic field was applied or not. This is evidenced by Figure 10, in which the results from the transfection and magnetofection experiments are summarized. 8198

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Figure 10. Flow cytometry measurements in the absence (transfection) and upon application of a magnetic field (magnetofection) evaluated by fluorescence and percentage of mean fluorescence of magnetopolyplexes at different P/N ratio: 1, cells; 2, Lipofectamine; 3, PEI (polyethylenimine p0.25); 4, p0.16; 5, p0.33; 6, p0.5; 7, p3. Right-hand axis gives the transfection efficiency relative to that of Lipofectamine, which is taken for 100%.

depending on the P/N ratio. Differences in the morphology of the initial magnetic micelles and the resulting magnetopolyplexes with either linear DNA or plasmid DNA were registered by TEM. Cytotoxicity screening of hybrid nanosystems in a spectrum of four human cell lines was undertaken as well. All vector systems are considered nontoxic as they did not induce any significant decrease in cell viability. Flow cytometry studies showed very high (about 45%) degrees of internalization into the cells, which was comparable and even higher than those of commercial transfection reagents such as Lipofectamine and dendritic poyethylenimine. The effect of the magnetic field on the cellular uptake was marginal. The transfection efficiency of the magnetopolyplexes was low (less than 60% the efficiency of Lipofectamine). The magnetic field, however, markedly boosted (up to 3.5 times) their transgene expression efficiency. Two of the magnetopolyplexes exhibited efficiency even higher than that of Lipofectamine, which was attributed to the proper balance between size, charge, DNA content, stability, and magnetic properties. The results indicated that the magnetic forces assisted the magnetopolyplexes to escape the endolysosomal pathway to a large extent, which was a substantial advantage over nonmagnetic delivery systems and spontaneous transfection.

superior efficiency reaching about 120 and 150%, respectively, the efficiency of Lipofectamine. The highest level of transgene expression was displayed by the complex p0.5, which apparently provided the most favorable balance between the particle dimensions, surface potential, DNA bioavailability (Table 2), stability, and magnetic properties. An obvious observation was that this complex was able to escape to the largest extent the endolysosomal pathway. At this stage of investigations it is not clear how. We can speculate on possible disruption of the membrane of the endolysosomal vesicles under the action of the oriented magnetic forces leading to subsequent release in the cytosol. Furthermore, the same forces may assist the diffusion of magnetopolyplexes in the cytosol thus overcoming the physical obstacles of the mesh-like structure of the cytoskeleton, which can severely impede the traffic to the nucleus. The high stability of the magnetopolyplex (data not shown) and the effective DNA packaging are beneficial to prevent the particles from nucleolytic enzymatic degradation in the metabolically hostile cytosolic environment; however, it might have limited the transgene expression.

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CONCLUSION Hybrid vector nanosystems for gene delivery were prepared by electrostatic interactions of PS-b-P2QVP-115K copolymer micelles loaded with magnetic nanoparticles and linear and plasmid DNA. Complete physicochemical and biopharmaceutical characterization of the hybrid vector nanosystems was performed. The magnetopolyplexes were prepared in a wide interval of P/N ratios ranging from 0.05 to 20. DLS measurements evidenced formation of well-defined magnetopolyplexes of narrow size distribution, particle dimensions below 360 nm, and ζ-potential varying from positive to negative

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Tel + 359 2 9792281; Fax + 359 8700309 (E.H.). Notes

The authors declare no competing financial interest. 8199

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ACKNOWLEDGMENTS This work was supported by the National Science Fund (Bulgaria) Project DMU 03/30/12.12.2012. We also thank the ESF research networking program Precision Polymer Materials and the EC project POLINNOVA. Dr. E. Haladjova expresses gratitude to UNESCO and L’Oreal fellowship program For Women in Science in Bulgaria.

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dx.doi.org/10.1021/la501402q | Langmuir 2014, 30, 8193−8200