Induction of Human Alveolar Epithelial Cell Growth Factor Receptors by Dendrimeric Nanostructures

Abstract

Although nonviral dendrimeric nanostructures have been widely used as gene delivery systems, key questions about target cells responses to these nanostructures are yet to be answered. Here, we report the responsiveness of A431 and A549 cells upon treatment with polypropylenimine diaminobutane (DAB) dendrimers nanosystems. Complexation of DAB dendrimers with DNA reduced the zeta potential of nanostructures, but increased their size. Fluorescence microscopy revealed high transfection efficiency in both cell lines treated with DAB dendrimers with induced cytotoxicity evidenced by MTT assay. The A549 cells showed upregulation of epidermal growth factor receptor (EGFR) and its downstream signalling biomolecule Akt kinase upon treatment with DAB dendrimers, while no changes were observed in A431 cells. Based on our findings, the biological impacts of these nanosystems appeared to be cell dependent. Thus, the biological responses of target cells should be taken into account when these nanostructures are used as gene delivery system.

To correct genetic disorders, genome-based therapeutics need to be successfully delivered to the target sites using an appropriate delivery system. The most challenging aim in gene therapy is advancement of safe and efficient gene carriers with appropriately high potential to encapsulate and deliver foreign genetic materials into the target cells and tissues. ¹ A gene delivery system ideally should pass through the biological membranes and barriers that selectively control cellular traverse ² and transfer the genomedicine with minimal impact to the integrity of target cells and tissues. Among the enormous number of research studies in gene delivery conducted worldwide, 2 paradigms of viral and nonviral vectors appear to be the main delivery approaches. Of these, many viruses such as adenovirus, retrovirus, herpes simplex virus, and pox virus have been modified to maximize transfection efficiency with minimal undesired biological impact. However, despite high transfection efficiency of viral vectors, ³ they have been reported to promote an inherent stimulation of the immune system that may carry serious consequences, as demonstrated by the death of a volunteer undergoing a clinical trial of ornithine transcarbamylase gene therapy. ^4,5 These safety and immunogenicity concerns, together with the limited capacity of transgenic materials, have directed researchers to increasingly focus on nonviral vectors, which are generally cationic in nature. ^1,6 They include cationic polymers, dendrimers, peptides, and liposomes. ^7–10 Although the nonviral vectors may provide lower transfection efficiency than the viral vectors, they are deemed to confer the advantages of safety, simplicity of preparation, and high gene encapsulation capability. ¹ Thus, they have received a great deal of attention as safer, alternative carriers to deliver nucleic acid–based therapies across biological membranes and barriers. ^11–18 Of nonviral vectors, cationic dendrimers possess appropriate architectural characteristics for condensation, incorporation, and delivery of gene therapies. For example, the lower generations of polypropylenimine diaminobutane dendrimers, DAB8 (G2) and DAB16 (G3), were reported as potential polymers to condense and carry such genome-based therapies into target cells. ¹⁹ Recently, we successfully exploited DAB dendrimers for delivery of antisense oligonucleotides to target epidermal growth factor receptor (EGFR) in human epithelial A431 cells, ²⁰ upon which we found that transfection potential of these gene delivery nanosystems was largely dependent on the type of target cells. To pursue cell-dependent biological impacts of DAB dendrimers, in the current study we investigated the expression of key biomarkers (ie, EGFR and its downstream molecule Akt kinase) in the human epithelial A431 and A549 cells.

Methods

Materials

Trireagent, DAB8, DAB16, ethidium bromide, isopropanol, chloroform, formaldehyde, dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)2,5- diphenyltetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), Triton X-100, and bovine serum albumin (BSA) were from Sigma-Aldrich Co (Poole, UK). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin G, streptomycin, L-glutamine 200 mM (×100), first-strand PCR buffer (×5), Moloney murine leukemia virus reverse transcriptase, dithiotheritol (DTT), and RNase/DNase free ddH₂O were purchased from Invitrogen (Paisley, UK). The molecular weight rainbow marker (10–250 kDa), deoxynucleotide triphosphate monomers (dNTPs), horseradish peroxidase (HRP), conjugated donkey anti-rabbit antibody NA934V and sheep anti-mouse antibody NA931V, and random hexamer primers (pdN6) were obtained from Amersham Biosciences (Bucks, UK). Rabbit anti-EGFR polyclonal antibody 2232 and rabbit anti-Akt antibody 9272 were purchased from Upstate Cell Signaling (Milton Keynes, UK). DC protein assay kit was from Bio-Rad (Hertfordshire, UK). Super Signal chemiluminescent substrate was from Perbio Science (Tattenhall, UK). Kodak autoradiography film was obtained from G.R.I. (Rayne, UK). Protran nitrocellulose membrane was from Schleicher & Schuell (Dassel, Germany). Tissue culture treated multiwell plates and flasks were obtained from Corning Costar (High Wycombe, UK). RNasin was from Promega (Southampton, UK) and Taq polymerase from Qiagen Ltd (Crawley, UK). FITC-labeled EGFR anti-sense was from MWG-Biotech (Ebersberg, Germany). DAPI (4′,6-Diamidino-2′-phenylindole dihydrochloride) was obtained from Roche (Leverkusen, Germany). Human epidermoid carcinoma A431 cell line and human alveolar adenocarcinoma A549 cell line were purchased from ECACC (Salisbury, UK). All other chemicals (not mentioned above) were from either Sigma (Poole, UK) or Fisher Scientific (Leicestershire, UK).

Cell Culture and Transfection

A431 or A549 cells were cultured at a seeding density of 5.0×10⁴ cells/cm² onto 6-well plates using normal culture medium (DMEM supplemented with 10% FBS, 100 units/mL penicillin G, and 100 μg/mL streptomycin). The 40% to 50% confluent cells were washed twice with serum free media (SFM) and then exposed to the prepared nanoparticles of polymers or polyplexes for 4 hours at 37°C incubation. After that, the cells were washed with SFM, replenished with normal culture medium, and incubated at 37°C for another 24 hours.

To prepare nanoparticles, a designated mass of DAB polymers alone (5, 10, 20 50, 100, 200 μg/mL) or complexed with a constant mass of salmon sperm DNA (4 μg/mL) or anti-EGFR antisense (4 μg/mL) was mixed initially in SFM by vortexing for 1 to 2 minutes and incubating at room temperature for 15 minutes. In accordance with studies conducted by Zinselmeyer et al, ¹⁹ a 5:1 ratio of DAB/DNA was used for the genocompatibility assessments.

Viability Assessment, Zeta Potential, and Particle Sizing of Polyplexes

To assess the influence of DAB dendrimers on cellular viability, the A431 or A549 cells were seeded and cultured up to 40% to 50% confluency in 96-well plates prior to treatment. The cultured cells were then exposed to a range of concentrations of polymers alone or polyplexes and incubated at 37°C for 4 hours. Next, the cells were washed once with phosphate-buffered saline (PBS), replenished with normal culture medium, and incubated at 37°C for 24 hours. The normal culture medium was replaced with 200 μL fresh media, and then 50 μL MTT reagent (2.5 mg/mL in PBS) was added to each well. Following a 4-hour incubation period at 37°C, medium was removed and the cells were exposed to 200 μL DMSO and 50 mL Sorenson buffer (pH 7.4). The cultures were incubated for 30 minutes at 37°C, and then ultraviolet absorbance was measured at 570 nm using a spectrophotometric plate reader, Anthos HtII, Anthos Labtec Instruments (Salzburg, Austria).

The zeta potentials of the DAB dendrimers alone or as complexed with DNA formed at various mass ratios were determined using microelectrophoresis with a Malvern ZetaSizer 3 (Malvern Instruments Ltd, Malvern, UK). The polyplexes were prepared using varying mass ratios (1:1, 5:1, and 10:1) of DAB polymer and DNA using distilled, degassed biologicgrade water. Samples were consecutively measured 10 times with instrument calibration (Malvern AZ55 Electrophoretic Standard) prior to each series of measurements. The DTS1050 was used as a standard control for negative charges. The same sample was then subjected to size analysis by photon correlation spectroscopy (Malvern ZetaSizer 3) using a 5-mW laser at an angle of incidence of 90°. This measurement was repeated 3 times in multimodal analysis. Both zeta potential and size determinations were performed at 25°C. Prior to use, all glass and plastic wares were prewashed with filtered water to minimize particulate contamination.

Fluorescence Microscopy

For fluorescence microscopy, cells were cultivated as explained onto the 22-mm² coverslips. At 40% to 50% confluency, they were transfected with an appropriate ratio of DAB/DNA complexes using FITC-labeled oligodeoxynucleotides (f-ODN) for 4 hours. The cells were washed 3 times with sterile PBS prior to fixation, which was performed by 10 minutes of incubation at room temperature using 2% formaldehyde in PBS. The cells were then washed 3 times with PBS and mounted on slides using mounting medium without or with DAPI (50 μM, for 20 minutes) for nuclear staining. The prepared samples were examined using an Olympus BX51 compound fluorescence microscope equipped with a BX-RFA fluorescence illuminator and catadioptric UMPlanFL-BD objectives. Intermediate magnifications were obtained using a U-CA magnifying device (Olympus Optical Co, Ltd, Tokyo, Japan), which was inserted between objective and camera. To optimize fluorescence excitation, a double-band fluorescence mirror unit, U-DM-DA/FI2 with excitation filter at 400 to 420 nm and 480 to 500 nm, was used for simultaneous observation of DAPI/FITC-stained samples. An alternative method was exploited using a U-MWU2 ultraviolet excitation cube at 330 to 385 nm for DAPI and a U-MWB2 cube at 460 to 490 nm for FITC-stained samples. To improve quality of the images, z-stacks acquisitions were performed (ie, by 5-μm increments per focal step) using Hamamatsu C7780-10 cooled CCD (Hamamatsu Photonic Co, Tokyo, Japan) as TIFF format in RGB mode (ie, 12 bits per channel). Acquired images (1344 × 1024 pixels resolution) were then imported to ImageJ 1.37 software (http://www.uhnres.utoronto.ca/facilities/wcif/imagej/) to produce the superimposed images. Depth of focus was improved automatically using the stack z-projection plug-in at the expert mode (complex wavelet).

Semiquantitative RT-PCR

To perform the reverse transcription-polymerase chain reaction (RT-PCR) analysis, total RNA was isolated from dendrimers treated (alone or as polyplexes) and untreated cells using Trireagent and examined for quantity and quality as described previously, and then a standard RT-PCR methodology was conducted using previously designed primers. ²¹ The PCR thermocycling program was as follows: denaturing at 94°C for 30 seconds, annealing at 57°C to 64°C for 45 seconds, and extension at 72°C for 45 seconds through a total of 30 cycles. The PCR recipe (for 25 μL reaction) consisted of 2.5 μL Taq 10× buffer, 1 to 3 μL MgCl₂ (25 mM), 2 μL dNTPs (5 μM), 2 μL complementary DNA (100 ng/1 μL), 0.25 to 1μL forward and reverse primers (10 pmol/1 μL), and 0.1 μL Qiagen Taq polymerase enzyme (5U/1 μL) and RNase/DNase-free ddH₂O (up to 25 μL). The PCR products were electrophoresed through a 1% agarose gel containing ethidium bromide (0.1 μg/1 mL) and visualized under ultraviolet light. The density of expressed bands was measured using a GS-700 densitometer and molecular analysis software (Bio-Rad, Hempstead, UK). The density of each spot was subjected to local background density subtraction and normalized to the density of the house keeping gene, β-actin. The ratio of treated over untreated samples was determined for 2 independent replicates.

Western Blot Analysis

Cells were grown to 40% to 50% confluency and exposed to reagents as described previously. Cells were washed 3 times with ice-cold PBS, and then total proteins were harvested using 100 to 200 μL lysis buffer (50 mM Tris, 5 mM EGTA, 150 mM NaCl, and 1% Triton) containing protease inhibitors (NaVO4, NaF, PMSF, phenylarsine oxide, sodium molybdate, leupeptin, and aprotinin). After 15 minutes of incubation on ice, cell debris was removed by centrifugation at 12,000 × g at 4°C for 15 minutes. The supernatant was analyzed for protein content using a standard BSA assay by means of DC protein assay kit (Bio-Rad, Hempstead, UK). Total protein concentration was measured at 750 nm using an ultraviolet/visible spectrophotometer, Ultraspec 3100 pro (Amersham Biosciences, Cambridge, UK). After mixing with loading buffer (60 mM Tris, pH 6.8, containing 2% [wt/vol] SDS, 10% [vol/vol] glycerol, 0.005% [wt/vol] bromophenol, and 250 mMDTT), the samples were boiled for 10 minutes at 100°C and equal amounts of protein (30 μg/lane) along with rainbow molecular weight marker were loaded onto an SDS–polyacrylamide gel electrophoresis (5% stacking gel and 8% resolving gel) and run at 100 V. Protein patterns were transferred onto Protran nitrocellulose membrane and the free sites were blocked using blocking buffer (5% [wt/vol] marvel dried skimmed milk [DSM] in the Tris-buffered saline [TBS]/Tween [0.05%]) for 2 hours at room temperature. Following blocking, the membrane was probed with designated antibodies:

EGFR: rabbit anti-EGFR antibody (1:1000 in TBS/Tween and 1% DSM) overnight incubation at 4°C, washing with 1% TBS (×5), followed by 2 hours incubation at room temperature with HRP-linked donkey anti-rabbit secondary antibody (1:10 000 in TBS/Tween and 5% DSM)

Akt: rabbit anti Akt antibody (1:1000 in TBS/Tween and 1% DSM) overnight incubation at 4°C, washing with 1% TBS (×5), followed by 2 hours incubation at room temperature with sheep anti-mouse secondary antibody NA931V (1:10 000 in TBS/Tween and 5% DSM)

β-actin: mouse monoclonal anti-β-actin antibody (1:20 000 in TBS/Tween and 1% DSM) overnight incubation at 4°C, washing with 1% TBS (×5), followed by 2 hours incubation at room temperature with sheep anti-mouse secondary antibody NA931V (1:10 000 in TBS/Tween and 5% DSM)

Immunoreactive bands were detected with SuperSignal chemiluminescent substrate using Kodak autoradiography film. Image acquisition and band quantification were conducted using a GS-700 densitometer with molecular analyst software (Bio-Rad, Hempstead, UK).

Results

The chemical structures of the polypropylenimine diaminobutane dendrimers are shown in Figure 1. Based on physicochemical properties, DAB 8 and DAB 16 possess 8 and 16 primary protonable surface amine groups, respectively, that indicate the capability of these polymers for DNA condensation.

MTT Survival Assay, Zeta Potential, and Size of Polyplexes

The cytotoxicity potential of the DAB dendrimers (alone or as complexed with DNA) was assessed in human epithelial cells using MTT survival assay. Upon treatment with DAB dendrimers, both A431 and A549 cells showed similar trends of cytotoxicity, which appeared to be dependent on concentration and generation of the polymers used (data not shown). DAB16 resulted in higher toxicity than DAB8 within both A431 and A549 cells. Figure 2 demonstrates cellular toxicity in both A431 and A549 cells after treatment with DAB16 (20 μg/mL/well) alone or as complexed with DNA. The cellular toxicity of DAB16 dendrimer was reduced in both cell lines after complexation with DNA (Figure 2). The polymer/DNA ratio of 5:1 and 10:1 (wt/wt) resulted in maximum reduction of cytotoxity.

To find possible correlations between surface zeta potential of and cytotoxicity induced by DAB dendrimers, the zeta potential of these polymers (alone or as complexed with DNA) was measured. The DAB8 and DAB16 dendrimers yielded zeta potentials of 10.3 ± 3.3 (mV) and 31.8 ± 8.0 (mV), respectively, which were significantly (P < .05) decreased to 1.9 ± 0.9 (mV) and 8.9 ± 2.4 (mV) upon complexation with DNA (Figure 3). The DAB/DNA polyplexes revealed a particle size distribution that provided mean diameters about 100 to 300 nm.

Fluorescence Microscopy

Fluorescently labeled ODN-DAB complexes were incubated with cells in serum-free medium, and the cell-associated fluorescence and subcellular distribution were assessed by fluorescent microscopy. DAPI was used to stain the nucleus (Figure 4A). The DAB dendrimers improved the cellular association of f-ODN in both A549 cells (Figures 4B and 4C) and A431 cells (Figure 4D). The cellular uptake of the naked f-ODN appeared to be very low (data not shown). The cytosolic and nuclear distribution of f-ODN revealed the gene delivery potential of DAB dendrimers to subcellular compartments, in particular the nucleus (Figure 4B).

RT-PCR and Western Blot Analyses

Figures 5 and 6, respectively, demonstrate the messenger RNA (mRNA) and protein expressions of EGFR within A431 and A549 cells after treatment with DAB dendrimers. The transcriptomes of EGFR showed no changes in A431 cells treated with DAB dendrimers (Figure 5A); however, these transcriptomes were up-regulated in A549 cells treated with DAB dendrimers alone or as complexed with DNA (Figure 5B). Similarly, upon use of DAB dendrimer alone or as a polyplex, the expression of the EGFR downstream signaling molecule Akt kinase was up-regulated in the A549 cells but not in A431 cells (Figure 5).

Western blot analysis was further undertaken to substantiate such a cell-dependent effect of DAB16 dendrimer on EGFR (Figure 6). Upon treatment with DAB dendrimers, overexpression of EGFR and Akt kinase was observed inA549 cells butnot inA431 cells.

Discussion

The transport of nucleic acids through the biological membranes and barriers is a prerequisite for gene therapy. An invasive traverse of gene-based nanoparticles is yet to be dependably accomplished despite some promising achievements using viral and nonviral vectors. Polycationic dendrimers, as potentially safer alternatives that are capable of condensing DNA to nanoparticles with radii of 20 to 100 nm, have been successfully used for gene delivery. ^22,23 These cationic structures are able to interact with negatively charged cell surface moieties; however, surprisingly little attention has been devoted to examining early or late impact of these structures on subcellular moieties in a cell/tissue-dependent manner despite increasing implementation of the bioactive nanomaterials. Hollins et al ²⁰ reported that the low generations of DAB dendrimers (ie, DAB 8 and DAB16) can efficiently deliver antisense oligonucleotides to target EGFR in human epithelial A431 cells. During this investigation, we perceived varied behavior from the tested cells. Thus, to pursue the biological influences of these polymers, we aimed to examine the effects of the DAB nanostructures on desired biomarkers (ie, EGFR and Akt kinase) in the human epithelial A431 and A549 cells.

Consistent with previous reports, ^19,20 our preliminary MTT assay resulted in cytotoxicity within epithelial cells after treatment with DAB8 and DAB16 dendrimers in a concentration- and generation-dependent manner. We observed that the cellular toxicity was mainly dependent on the cell type and polymer generation; that is, DAB dendrimers induced greater cytotoxicity within the A549 cells than within the A431 cells. In addition, the DAB16 dendrimer generated more toxicity than DAB8 within both cell lines. Once these dendrimers were complexed with DNA, slight (but not significant) upturns were observed in both cell lines (Figure 2). We speculate that such diminished cytotoxicity could be a result of the decreased zeta potential of nanostructures, by which the interaction of dendrimers with the negatively charged elements of cell membrane could be weakened. Surface modification of polymeric vectors can alter its cytotoxic potential. For instance, surface modification of the cationic starburst polyamidoamine (PAMAM) dendrimers with either lauroyl chains or polyethylene glycol 2000 was reported to reduce the cytotoxicity of these dendrimers in Caco- 2 cells because of reduction and/or shielding of the dendrimers surface positive charge. ²⁴ Further, Zinselmeyer et al ¹⁹ reported enhanced cellular toxicity of dendrimers in association with molecular size (ie, the larger the molecular size, the greater the cytotoxicity). This is perhaps attributable to the availability of multiple contact points between dendrimer molecules and cell surface elements. It has been shown that the cytotoxic impact of polycationic paint components (ie, FWR, a polyurea; FWN, a polyamide-amine; FHN, a polyamine) in cell cultures (eg, primary cultures of rat and human type II pneumocytes; alveolar macrophages and human erythrocytes) can be markedly diminished in the presence of the polyanions. ²⁵ Significant reduction in toxicity of the DAB dendrimers has been reported following the removal of some of the available anion binding sites by complexation with DNA. ¹⁹ In addition to surface charges of polycations, the structural architecture of cationic materials may play an important role in the cellular toxicity. ^26–28 A few researchers have also reported that the complexation of some polymers (eg, Superfect, ExGen 500, Polyethylenimine, PEI 25 and 50) with DNA may increase cellular toxicity. ²⁷

Because the concentration ratio of a cationic polymer and DNA as polyplex nanostructures has been well recognized as a critical parameter for optimization of transfection, ^29–32 we used a ratio of 5:1 (wt/wt) of polymer/DNA in our work. This ratio, as previously reportedby Zinselmeyer et al, ¹⁹ can result in high transfection efficiency with minimal toxicity. We found that the zeta potential of the DAB dendrimers was diminished dramatically upon complexation with DNA, resulting in DAB/DNA nanoparticles (∼200–300 nm in diameter) as demonstrated in Figure 3.

Fluorescence microscopy analyses revealed profound distribution of the f-ODN/DAB nanoparticles in the cytosol and nucleus of both cell lines (Figure 4), whereas the FITC-labeled DNA alone failed to show significant internalization. This clearly implies that dendrimers possess the capability to circumvent plasma membranes and directly enter the nucleus compared with the naked f-ODN as reported previously. ²⁰ Despite the potential of DAB dendrimers for gene therapy, it appears that the biological interactions between polymer and cellular elements may induce some inadvertent consequences, which need to be fully studied. Based on our previously reported investigation using microarray technology, the DAB16/DNA nanoparticles induced some gene expression changes in A549 cells despite a reduced phenotypic cellular toxicity (obtained from MTT), perhaps attributable to the reduction of surface charge of DAB16/DNA nanoparticles. We found expression changes for some important genes (ie, tgfβ1, bcl2α1, il5, cxcr4, and pckα) within A549 cells. ³³ Among these, for example, the up-regulation of tgfβ1 and bcl2αcan conceivably imply the occurrence of apoptosis upon treatment with DAB16/DNA polyplexes. When DAB dendrimers were used for delivery of the anti-EGFR ODN in human alveolar epithelial A549 cells (data not shown), we observed little effect of the nanogenomedicine on downregulation of EGFR. These results led us to examine the effect of DAB dendrimers themselves on the expression of EGFR for both transcriptomic and protein levels. Upon use of dendrimers, significant up-regulation of EGFR (mRNA and protein) was observed within A549 cells (Figures 5 and 6, respectively) but not in the A431 cells. Presumably, either these cells are more sensitive than A431 cells because of differences in cellular characteristics or the expression of EGFR in A431 cells is at a level of saturation. Our preliminary examinations showed that the expression rate of EGFR in A431 cells is greater than that in A549 cells. Because the DAB dendrimers impose cytotoxicity in both cell lines (Figure 2), we speculate that the cells fight back against such xenobiotic stresses to survive. As a result, they induce the expression of EGFR and accordingly Akt kinase. However, the A549 cells need to induce higher amounts of EGFR compared with the A431 cells. The DAB/anti-EGFR ODNs nanosystems were shown to reduce the expression of EGFR in A431 cells, ²⁰ whereas the DAB dendrimers alone resulted in up-regulation of EGFR and Akt kinase 1 in A549 cells. This clearly implies that the gene-targeting strategies should be based on the biological characteristics and impacts of the target cells and tissues. The EGFR downstream molecule Akt kinase 1 promotes cell survival by inhibiting cell death activators and in some cases by inhibiting the transcription of genes that encode them. ^34,35 The serine/threonine protein kinase Akt/PKB is the cellular homologue of the viral oncogene v-Akt that is activated by a variety of growth and survival factors. There exist 3 identified isoforms of the Akt kinase (ie, Akt1, Akt2, and Akt3) in mammals. Various cell surface receptors may elicit the production of second messengers that can activate phosphoinositide 3-kinase (PI3K). Akt is located downstream of PI3K, which is a downstream signaling molecule elicited by activation of EGFR. ^36,37

It appears that the cytogenomic toxicity induced by DAB16 dendrimer or DAB16/DNA nanoparticles may be attributed to the structural characteristics of the polymer. Such a phenomenon was also observed in A431 cells treated with lipid-based gene delivery systems ^21,38 or with the starburst PAMAM dendrimers. ³⁹

Given our previous investigation on genocompatibility of cationic lipids within target cells, ²¹ together with the results obtained in the current study, it appears that the cationic gene delivery DAB dendrimer nanosystems may inadvertently elicit the expression of EGFR and Akt kinase in A549 cells but not in A431 cells. These findings indicate that the biological impact of polycationic dendrimers seems to be cell dependent. Thus, to avoid any undesired influences of the cationic gene delivery nanosystems, we suggest that the biological responses of target cells be taken into account when transfected with the cationic nanosystems.