DEVICE, METHOD AND COMPOSITION FOR TRANSFECTION OF CELLS WITH NUCLEIC ACIDS

Abstract

Disclosed are a method, a composition and a device for the introduction of exogenous nucleic acids into eukaryotic cells by non-viral vectors (non-viral transfection). The method according to the invention is based on application of a high-frequency oscillatory motion to a solution containing nucleic acids and cationic polymers or lipids to obtain particles (complexes) with high transfection efficiency.

Claims

1. A method for preparing a composition for non-viral transfection of cells with nucleic acids, said method comprising applying an oscillatory motion with frequency ranging from 100 Hz to 10 kHz and oscillation amplitude ranging from 200 nm to 2 mm, to a solution containing, in uncomplexed form, a cationic polymer or lipid and the nucleic acid of interest.

2. The method according to claim 1, which comprises the following steps: (i) providing, in a suitable container, an aqueous solution containing a cationic polymer or lipid and the nucleic acid of interest; (ii) applying an oscillatory motion with frequency ranging from 100 Hz to 10 kHz and oscillation amplitude ranging from 200 nm to 2 mm to said container.

3. The method according to claim 1, wherein the oscillatory motion is uniaxial.

4. The method according to claim 1, wherein the oscillatory motion is along the vertical axis.

5. The method according to claim 1, wherein the oscillatory motion has a sine, square or triangular waveform.

6. The method according to claim 1, wherein the oscillatory motion is applied for a time ranging from 1 sec to 30 min.

7. The method according to claim 1, wherein the cationic polymer is selected from linear or branched polyethylenimine (lPEI and bPEI), polyamidoamine (PAMAM), D- or L-polylysine (PDL and PLL), chitosan, poly(dimethylaminoethyl methacrylate) (PDMAEMA) and poly(diethylaminoethyl methacrylate) (PDEAEMA).

8. The method according to claim 1, wherein the cationic lipid is selected from 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), dioctadecylamidoglycylspermine (DOGS), dimethyl-dioctadecylammonium bromide (DDBA), 3□-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-chol), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristoyloxy-propyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE), and cetyltrimethyl-ammonium bromide (CTAB).

9. The method according to claim 2, wherein the container is a test tube having a capacity ranging from 0.1 mL to 50 mL.

10. The method according to claim 1, wherein the frequency of the oscillatory motion ranges from 100 Hz to 1 kHz.

11. The method according to claim 1, wherein the amplitude of the oscillatory motion ranges from 200 nm to 0.2 mm.

12. (canceled)

13. A method for cell transfection in a cell culture, which comprises contacting the cell culture with a composition according to claim 12.

14. The method according to claim 13, which comprises the following steps: (i) providing a cell culture containing a culture medium; (ii) adding to the culture medium a composition for non-viral cell transfection obtainable with a method comprising applying an oscillatory motion with frequency ranging from 100 Hz to 10 kHz and oscillation amplitude ranging from 200 nm to 2 mm, to a solution containing, in uncomplexed form, a cationic polymer or lipid and the nucleic acid of interest.

15. A device for implementing the method of claim 1, which comprises a generator of waves having the required frequency and amplitude, and an electromechanical actuator provided with a drive shaft having a test tube housing at one end, whereby the voltage produced by the generator is converted by the actuator into an oscillatory motion which is transmitted to the test tube via the drive shaft.

16. The method according to claim 2, wherein the container is a test tube having a capacity ranging from 0.1 mL to 1.5 mL.

17. The method according to claim 1, wherein the frequency of the oscillatory motion is equal to 1 kHz.

Description

DESCRIPTION OF FIGURES

[0034] FIG. 1: Scheme of device used; sine, square or triangular wave generator with controlled amplitude and frequency (1), mechanical actuator that converts the input voltage into motion along z-axis (2), laboratory test tube housing which transmits the oscillatory uniaxial motion to a test tube (3).

[0035] FIG. 2: Results, expressed in terms of cytotoxicity (A, C) and transfection efficiency (B, D), obtained in vitro on L929 and HeLa cell lines and on bovine chondrocytes (primary cells) with complexes based on plasmid DNA (pGL3) and the cationic polymer polyethylenimine (PEI); A, B) PEI in branched form; C, D) PEI in linear form obtained by the method according to the invention and standard preparation; E) Results expressed in terms of increased transfection efficiency of complexes based on plasmid DNA (pGL3) and PEI (in branched form (bPEI) and linear form (lPEI)) prepared by the method according to the invention vs. the standard preparation. The data are expressed as mean ±standard deviation) (n≥3). *p<0.05.

[0036] FIG. 3: Cytotoxicity and transfection efficiency on L929 cells of (i) a composition according to the invention obtained by varying the mixing frequency of cationic polymer and nucleic acid, (ii) a standard preparation obtained by manual mixing, and (iii) a commercial kit (jetPEI®). The data are expressed as mean±standard deviation (n≥3). *p<0.05 vs. standard preparation.

[0037] FIG. 4: Cytotoxicity and transfection efficiency on L929 cells of (i) a composition according to the invention obtained by vertical mixing of cationic polymer and nucleic acid using various waveforms, (ii) a standard preparation obtained by manual mixing, and (iii) a commercial kit (jetPEI®). The data are expressed as mean ±standard deviation (n≥3). *p<0.05 vs. standard preparation.

[0038] FIG. 5: Cytotoxicity and transfection efficiency on L929 cells of (i) a composition according to the invention obtained by vertical and horizontal mixing of cationic polymer and nucleic acid, (ii) a standard preparation obtained by manual or vortex mixing, and (iii) a commercial kit (jetPEI). The data are expressed as mean±standard deviation) (n≥3). *p<0.05.

[0039] FIG. 6: Transfection efficiency on L929 cells of (i) a composition according to the invention and (ii) a standard preparation in the absence or presence of cell stimulation with high-frequency vertical oscillatory uniaxial motion. The data are expressed as mean±standard deviation) (n≥3). *p<0.05.

EXAMPLE 1—DEVICE

[0040] A bench prototype device was assembled, consisting of an electromechanical actuator or mechanical wave driver (component 2 in FIG. 1) which converts input voltage into uniaxial motion of a drive shaft. Said component is coupled to a sine, square or triangular wave generator (component 1 in FIG. 1), which allows its control in terms of frequency (frequency range: 100 Hz-10 kHz) and amplitude of motion (displacement range: 200 nm-2 mm). The optimum conditions of use are: i) input wave type: sine wave; ii) direction of motion: vertical (z-axis); iii) frequency: 1 kHz, iv) displacement: 200 nm. Both components are marketed by Arbor Scientific (https://www.arborsci.com/) as demonstrators for toys or teaching purposes for the production of harmonic waves. However, other devices already available on the market or developed ad hoc, having a test tube housing for the preparation of different transfection compositions in parallel, can be used. In the prototype used, the shaft of the mechanical actuator was suitably fitted with a laboratory test tube housing (component 3—FIG. 1), to which it integrally transmits the drive shaft motion and allows controlled mixing of the transfection reagents contained in the laboratory test tube. In particular, the application of high-frequency oscillatory motion along the z-axis allows controlled mixing of cationic polymers or lipids and nucleic acids, thus producing nanoparticles and microparticles or transfection complexes in a reproducible, controlled way (FIG. 1). The oscillatory motion can be suitably varied in terms of displacement and frequency of oscillation of the test tube.

EXAMPLE 2—PREPARATION OF TRANSFECTION COMPOSITION

[0041] (i) A solution of 25 kDa cationic polymer polyethylenimine (PEI, in its linear form (lPEI) and branched form (bPEI)) was prepared at a concentration of 0.86 mg/mL, corresponding to an amine concentration ([N]) of 20 mM, in a 10 mM HEPES buffer solution at pH 7.5. Alternatively, the cationic polymer can be dissolved in deionised water (dH.sub.2O); saline solution (150 mM NaCl; pH=7.5); HGB (10 mM HEPES+5% (weight/volume) glucose; pH 7).

[0042] The solution was stored at 4° C. until use.

[0043] (ii) A solution of plasmid DNA containing the gene that encodes for the intracellular protein luciferase (pGL3-Control Vector, 5.2 kbp) was prepared separately at a concentration of 0.25 μg/μL in sterile aqueous solution 0.1x TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). If siRNA, miRNA, shRNA or mRNA is used, the nucleic acid can be dissolved in sterile 1×siMAX buffer (6 mM HEPES, 20 mM KCl, 0.2 mM MgCl.sub.2, pH 7.3) (Eurofins Genomics, Germany) at a concentration of 0.1 μg/μL.

[0044] The solution was stored at −20° C. until use.

[0045] (iii) All reagents were heated to the desired temperature before use.

[0046] A preselected volume of cationic polymer solution was transferred to a test tube to which a preselected volume of pDNA solution was added in a 10:1 volumetric ratio (v/v). Specifically, an amount of polymer containing a number of moles of amines such as to form polyplexes with an N/P ratio (ie. the ratio between the number of moles of amines of the cationic polymer and the number of moles of phosphates of the nucleic acid) of 30 was used.

[0047] (iv) The test tube was inserted into the housing suitably connected to the shaft of the mechanical actuator of the device (FIG. 1).

[0048] (v) The wave generator was operated at a mixing frequency of 1 kHz for 10 sec.

[0049] (vi) The resulting solution was immediately administered to the cells previously seeded in the culture plate.

EXAMPLE 3—PREPARATION OF TRANSFECTION COMPOSITION AT VARIOUS FREQUENCIES

[0050] The process was conducted as in the previous example, steps (i)-(iii).

[0051] The solution obtained in step (iii) was mixed manually with a micropipette for 10 sec and incubated for 20 min.

[0052] Alternatively, the solution was mixed by the method according to the invention at various mixing frequencies (100 Hz, 500 Hz, 1 kHz and 4 kHz) for 10 sec.

[0053] The resulting solution was administered to cells previously seeded in the culture plate, as described in the example 6.

EXAMPLE 4—PREPARATION OF TRANSFECTION COMPOSITION USING VARIOUS WAVEFORMS

[0054] The process was conducted as in the previous example, steps (i)-(iii).

[0055] The solution obtained in step (iii) was mixed manually with a micropipette for 10 sec and incubated for 20 min.

[0056] Alternatively, the solution was mixed by the method according to the invention. The waveform of the generator was suitably set as a sine, square or triangular wave, and the generator was operated at a stimulation frequency of 1 kHz for 10 sec.

[0057] The resulting solution was administered to cells previously seeded in the culture plate, as described in the example 6.

EXAMPLE 5—PREPARATION OF COMPARATIVE STANDARD

[0058] The process was conducted as in the example 2, steps (i)-(iii).

[0059] The solution obtained in step (iii) was mixed manually with a micropipette for 10 sec and incubated for 20 min.

[0060] Alternatively, the solution was mixed with a vortex.

[0061] The resulting solution was administered to cells previously seeded in the culture plate.

EXAMPLE 6—TRANSFECTION EXPERIMENTS

[0062] Cell lines L929 (murine fibroblasts from subcutaneous connective tissue, CCL-1) and HeLa (human epithelial cells from ovarian cancer, CCL-2) were purchased from the American Type Culture Collection (ATCC®, Manassas, Va., USA). The primary chondrocytes were obtained from bovine metacarpophalangeal joint as previously described [Candiani G. et al., Chondrocytes response to high regimen of cyclic hydrostatic pressure in 3-dimensional engineered constructs, Int. J. Artif. Organs. 2008, 31(6), 490-499]. The cells were seeded in 96-well culture plates at a cell density of 2×10.sup.4 cells/cm.sup.2 and cultured at 37° C. in a humidified atmosphere and 5% (volume/volume) CO.sub.2 (hereinafter called “standard culture conditions”) in culture medium (Dulbecco's Modified Eagle's Medium (DMEM) completed with 10% (volume/volume) foetal bovine serum (FBS), 10 mM HEPES, 100 U/L penicillin, 0.1 mg/mL streptomycin, 1 mM sodium pyruvate and 2 mM glutamine (hereinafter called “complete medium”). 24 hours after seeding, 160 ng/cm.sup.2 of pGL3 (in the case of cell lines) and 320 ng/cm.sup.2 of pGL3 (in the case of primary cells) were complexed in 10 mM HEPES buffer with lPEI or bPEI using the method according to the invention or standard preparation as described in examples 2-4 and 5, respectively. Complexes prepared with a commercial kit (ie. jetPEI®) were obtained by mixing 780 ng/cm.sup.2 of pGL3 with the commercial transfectant solution by the procedures reported by the supplier and used as internal reference. The complexes were dispensed to the cells in 100 μL/well of complete medium, and the cells were incubated for 24 hours under standard culture conditions.

[0063] To compare the invention with the state of the art, L929 cells were treated with polyplexes obtained by the method of invention and by standard preparation, respectively, and they were subsequently subjected to mechanical stimulation with vibrations for 5 min at 1 kHz.

[0064] The cytotoxicity of the polyplexes was evaluated 24 hours after transfection by Alamar Blue® assay. Specifically, the medium in each well was eliminated and replaced with 100 μL/well of complete medium containing 1:9 (volume/volume) of resazurin. The cells were incubated for 2 hours under standard culture conditions, and the fluorescence was then read with a Synergy H1 multiplate reader (BioTek, Italy), using as excitation and emission wavelengths λ.sub.excitation=540 nm and λ.sub.emission=595 nm respectively. Cytotoxicity was calculated as follows:

cytotoxicity (%)=100%−viability (%)

[0065] wherein 100% was assigned to the untransfected cells (CTRL).

[0066] The transfection efficiency was determined by measuring the luciferase activity. Specifically, after being washed with Phosphate Buffered Saline (PBS), the cells were lysed with 110 μL/well of lysis solution (Promega, Italy) and subjected to freezing-thawing cycles to promote lysis.

[0067] 20 μL of cell lysate was then mixed with 50 μL of Luciferase Assay reagent (Promega, Italy) to measure luciferase activity. The chemiluminescence signal (Relative Light Unit, RLU) of each sample was acquired with a Synergy H1 multiplate reader and normalised to the total protein quantity, calculated by BCA assay (ThermoFisher, Italy). Transfection efficiency was expressed as RLU/mg of protein. Each experiment was conducted at least in triplicate (n≥3), and the data are expressed as mean±standard deviation.

[0068] The results shown in FIGS. 2-5 clearly indicate that the composition according to the invention: (1) has greater transfection efficiency than the standard preparation obtained by manual or vortex mixing, (2) exhibits lower toxicity than the commercial kit, and (3) is effective on different types of cell lines as well as on primary cells.

[0069] Using the same experimental model, the system according to the invention was also compared with the standard method applied to cell cultures in the presence or absence of cell stimulation with high-frequency oscillatory motion. As illustrated in FIG. 6, polyplexes obtained by the method of invention exhibit high transfection efficiency regardless of the presence of mechanical-vibrational cell stimulation, whereas with the standard preparation, comparable efficiency to that of the method of invention is only obtained with mechanical-vibrational stimulation of the cells. This result demonstrates that unlike the standard preparation, the system according to the invention guarantees high transfection efficiency with no need to pre-treat or stimulate the cells, thus avoiding additional toxicity problems or cell phenotype modifications.