TEST OF COMPLEX NANOSTRUCTURES AS GENE DELIVERY SYSTEM TO CARDIAC CELLS

Various nanostructures, including liposomes, dendrimers, polymers, carbon or silicon materials, and metal nanoparticles seems to be evaluated as carriers in different gene delivery systems. Muscle cells are target of modern gene medicine, due to of the much pathology (arrhythmias, channels pathological mutation, abnormal apoptosis) with limited curability by traditional drugs. The zeta potential of the muscle cells were identified by non-traditional method of DLS. This was starting point to setting of parameters for experimental liposome, the liposome surface charge have been tested and modified by cationic plasmid and final complex was evaluated as delivery system of DNA into the muscle cardiac cells.


INTRODUCTION
The zeta potential of the cells and the zeta potentials of the modern "Trojan horse particles" (liposomes, exosomes, nanoparticles) are two key parameters, which must be tested to ideal compatibility, if there is the aim of the maximal effectivity of "Trojan soldiers" homing into the living cells. The DLS technique was previously applied primarily in order to study microorganisms, cancer cells or blood cells [1,2]. We used a Zetasizer Nano ZS analyser to analyse the zeta potential of cardiomyocytes and also for characterisation of nanoparticles/nanoliposomes conjugated with GFP vector, which can be used s delivery system into the muscle and another type of mammalian cells. The analysis was correlated also with tests based on electrophoresis methods. Transfection of the cells and gen/protein delivery is the modern direction of the experimental medicine, however precise description of this phenomenon and developing of new "nano-entities" need precise analysis of the surface charge and precise analysis of the interaction between cells and selected "Trojan particles".

Preparation of two types of cardiac cells
Neonatal rat cardiomyocytes were isolated from Wistar 2-5 days old Neonatal rats. Hearts were removed and dissociated in a balanced salt solution containing 20 mM HEPES, 120 mM NaCl, 1 mM NaH2PO4, 5.5 mM glucose, 5.4 mM KCl, and 0.8 mM MgSO4 (pH 7.3-7.4). Cardiomyocytes from the tissue homogenates were isolated by trypsin digestion, selection of cardiomyocytes were done using adhesive affinity to collagen bottom described in our previous experiments [3]. The cardiomyocytes cultures were suspended in DMEM and transferred (1000 cells/ 100 µL) to 96 well culture chamber plates, which was rewarmed to 37°C. The first part of the cell culture was resuspendend and immediately taken from the bottom of plastic chamber and used for Zetasizer measurement of surface charge (chapter 2.2). The second part of the cells was incubated in DMEM medium and used for transfection tests (chapter 2.4) on microscopic unit.
Adult rat cardiomyocytes were obtained from young adult Wistar rats (250 ± 50 g). The cardiomyocytes were isolated using the method of enzymatic dissociation via [4] (the heart was perfused with Ca 2+ free Tyrode's solution containing collagenase, the final dissociated cardiomyocytes were collected in Ca 2+ free Tyrode's solution and gently mixed with standard Tyrode's solution (0.09 mmol/l CaCl2) and diluted 1:1 byDMEM medium in 96 well chamber in following 30 minutes. The first part of the cell culture were resuspendend from the bottom of plastic chamber after 10 minutes and used for Zetasizer measurement of surface charge (chapter 2.2). The second part of the cells was used for transfection tests (chapter 2.4). All experiments underwent under improvement of ethics committee.

The cell surface charge identification by Zetasizer
The zeta potential of the cells was recorded in a suspension (0.3 × 10 6 cells/ml) using the electrophoretic light scattering technique on a Zetasizer Nano ZS analyser (Malvern Instruments, Great Britain). The measurements were performed in a U-shaped cell with gold-plated electrodes at 25°C or 37°C.
Maghemite nanoparticles (FeNP) were synthesized by borohydride reduction (53 mmol in liquid ammonia) of ferric chloride FeCl3·6H2O (37 mmol), both purchased from Sigma-Aldrich Co. (St Louis, MO, USA), at room temperature. After the reduction reaction (indicated by change of colour to black), the temperature of the mixture was increased to 100°C and held constant for 2 hours. After cooling the mixture to room temperature, the magnetic fraction was separated using an external magnet and washed several times with water. Details in [5].
The surface of the liposomes or nanoparticles was modified by addition of plasmid pcDNA4/TO-bio-myc-NES-EGFP to form final DNA-particle of DNA-liposome complexes. Conjugation was tested with three different concentration of plasmid (10 μg/μL, 20 μg/μL, 40 μg/μL) in conjugation solution (timing and temperature described in Results) and after the conjugation step the electrophoresis was used for quantitative detection of amount of free (non bound) plasmid and relative effectivity of plasmid binding (details in Results).

The cell surface charge identification by Zetasizer
The zeta potential of cardiomyocytes in DMEM medium was measured under 25°C or 37°C respectively; all variants are reprinted in the following Table 1.

The nano-complexes surface charge identification by Zetasizer and selection of the optimal concentration of solution for conjugation
The   The surface charge detected in DMEM medium by Zetasizer was -5.3 mV for plasmid-FeNP complex (variant 20ng/μL) and -1.1 mV for plasmid-NL complex (the variant 20 ng/μL)

The test of the final transfection effectivity on the live cells
Both types of optimized "Trojan horses" (complex based on liposomes or nanoparticles) were tested on cell cultures (addition of previously prepared plasmid-particle complex to DMEM at 37°C and 5% CO2 atmosphere of the lab incubator, absolute plasmid-particle concentration in solution was adjusted to 0,8 ng/μL in all finally comprised variant). The final GFP positive cells (48 hours after transfection) are describe in Table 2, the representative microscopically scans of cells are on Figure 3.  The very actual preliminary data also illustrated the possibility of higher effectivity of invasion NL or FeNP complexes to the cardiac cells, if the altering electromagnetic field is applied on the cell samples (data not shown), optimizing of the electromagnetic field and its biocompatibility with muscle cells and covering tissue was stared in our parallel project [1].

CONCLUSION
Our investigation has two basic outputs. The complex zeta potential analysis of cells and entities for gene delivery (cardiomyocytes and nanoparticles or nanolyposomes) has been identified using zetapotential mode of standard DLS machine. The amount of the bound DNA on final "Trojan nanosystems" and charge profile of these nano-systems was adjusted and the optimised variant was used for pilot transfection experiments on cardiac cells. Transfection effectivity was 5-20%, however the methods have potential to be upgraded by additive electromagnetic field which accelerate invasion of charged complex into the cells.