Fluorinated cationic polymer, preparation method and application of fluorinated cationic polymer in gene transfection
By fluorinating poly(2-hydroxypropyleneimine), a fluorinated cationic polymer PHP-FH is formed, which solves the cytotoxicity and stability problems of existing vectors, and achieves efficient and low-toxicity gene transfection, suitable for transfection of various cell types.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-03
- Publication Date
- 2026-03-10
AI Technical Summary
Existing cationic polymer gene delivery vectors such as PEI have problems with high cytotoxicity and biological environmental sensitivity, which affect their application range and stability. In particular, they are prone to deterioration in aqueous solutions, making it difficult to achieve efficient and low-toxicity gene transfection.
Fluorination of poly(2-hydroxypropyleneimine) with fluorinated ethylene oxide compounds forms a fluorinated cationic polymer PHP-FH. By introducing polyfluoroalkyl chains, the stability and hydrophobic and lipophilic properties of the complex are enhanced, the positive charge density is reduced, and the biocompatibility and cell absorption efficiency are improved.
It significantly reduced cytotoxicity, improved gene transfection efficiency and the stability of the complex in serum, achieving highly efficient and low-toxicity in vitro and in vivo gene transfection, and has broad clinical application potential.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of polymer gene transfection carrier, and relates to a fluorinated cationic polymer, a preparation method and application thereof in gene transfection. BACKGROUND
[0002] It is crucial to construct safe and high transfection efficiency non-viral gene delivery vectors to improve the effectiveness of gene delivery for cancer treatment. Common non-viral vectors mainly include liposomes, polymer carriers and nanoparticle carriers. Part of cationic polymers have rich positive charges and special spatial structures, which can form stable complexes with DNA or RNA to promote endogenous or exogenous transfection of genes. By modifying the existing cationic polymer gene delivery vectors, the transfection performance, cytotoxicity, immunogenicity, loading capacity and targeting of the vectors can be effectively improved.
[0003] Cationic polymers, such as polyethyleneimine (PEI), are widely used due to their high transfection efficiency and good gene protection ability, but their high cytotoxicity and sensitivity to biological environment limit their application range. Researchers have tried many methods to reduce the cytotoxicity of the materials and improve the stability of the transfection reagents. Yuan Weien et al. modified PEI material with fluoralkyl chains by using heptafluorobutyric anhydride, so that PEI had hydrophobic and oleophobic properties, and the serum stability of the complex was improved (Yuan, Z. H.;Guo, X. S.;Wei, M. Y.;et al, Novel fluorinated polycationic delivery of anti-VEGF siRNA for tumor therapy. Npg Asia Materials 2020, 12 (1).). Guan Zhabin et al. functionalized low molecular weight PEI (Mw = 600) with fluorinated carbon epoxides of different lengths, so that the complex formed by the polymer and genetic material showed higher cell uptake and stronger serum stability (Zaliauskiene, L.;Bernadisiute, U.;Vareikis, A.;et al, Efficient Gene Transfection Using Novel Cationic Polymers Poly(hydroxyalkylene imines). Bioconjugate Chemistry 2010, 21 (9), 1602-1611.).
[0004] Poly(2-hydroxypropylene imine) (PHP) materials have a wide range of hydroxyl groups, better hydrophilicity than polyethylene imine, better biocompatibility, lower cytotoxicity, better stability of the gene delivery system formed after loading genes in serum, and improved gene transfection efficiency (Zaliauskiene, L.; Bernadisiute, U.; Vareikis, A.; et al, Efficient Gene Transfection Using Novel Cationic Polymers Poly(hydroxyalkylene imines). Bioconjugate Chemistry 2010, 21 (9), 1602-1611.). Through extensive exploration, it is found that the fluorination strategy is suitable for PHP materials, and fluorinated modification of poly(2-hydroxypropylene imine) with perfluoro fatty acid anhydride can realize efficient and low-toxicity transfection of various cells in vitro, and efficient and low-toxicity local administration in vivo, but the stability of the material in aqueous solution is poor, and deterioration often occurs during solution storage (Chen Dafeng; Li Kun; Bai Xin. A fluorinated cationic polymer gene carrier and a preparation method thereof: CN202311834202.1.2023.12.28). SUMMARY
[0005] The application provides a fluorinated cationic polymer, a preparation method thereof, and application thereof in gene transfection. The fluorinated cationic polymer poly(2-hydroxypropylene imine) (PHP) is obtained by fluorination of an oxirane fluorine-containing compound, and a high-efficiency and low-toxicity fluorinated cationic polymer gene transfection carrier PHP-FH is obtained.
[0006] The fluorinated cationic polymer has the following structural formula:
[0007]
[0008] In the formula, x is an integer greater than 0, y is an integer greater than or equal to 0; R is H or a polyfluoroalkyl chain, and part of R is not H, and the polyfluoroalkyl chain is selected from one of the following:
[0009]
[0010] Preferably, the molecular weight of the fluorinated cationic polymer is 3500-13000.
[0011] The preparation method of the fluorinated cationic polymer is as follows:
[0012]
[0013] The specific steps are as follows:
[0014] The PHP solution is added with an ethanol solution of an oxirane fluorine-containing compound, and the reaction is stirred at room temperature. After the reaction is completed, the product is dialyzed in ethanol and water in sequence, and vacuum freeze-drying is performed to obtain the fluorinated cationic polymer PHP-FH, and the oxirane fluorine-containing compound is selected from one of the following:
[0015]
[0016] Preferably, the molar ratio of PHP and the oxirane fluorine-containing compound is 1:(30-100).
[0017] Preferably, the stirring reaction time is 2-3 days.
[0018] Preferably, the specific dialysis method is: first dialyzed in ethanol for 2-4 days through a cellulose membrane with a pore size of 3500 Da or above, and then dialyzed in water for 2-4 days.
[0019] Preferably, the vacuum freeze-drying method is: first pre-frozen at -80℃ for 1 day, and then vacuum freeze-dried for 2-4 days.
[0020] The application provides the use of the above fluorinated cationic polymer as a gene transfection carrier in gene transfection.
[0021] Preferably, the mass ratio of the fluorinated cationic polymer and the loaded gene is (1-50):1.
[0022] Preferably, the gene is DNA, siRNA, shRNA, microRNA or modified nucleic acid.
[0023] The application links fluorinated alkyl to PHP through oxirane ring-opening reaction, grafts polyfluoroalkyl chains on the surface of PHP by fluorination modification of PHP, can enhance the stability of the polymer / DNA complex by introducing forces other than ionic bonds in the polymer / DNA complex, at the same time, ensures the stability of the complex in the biological membrane environment. The fluorinated chain combines the characteristics of hydrophobicity and lipophobicity, can be combined with each other through fluorophilic effect, and can also be assembled with genes. At the same time, the fluorinated polymer reduces the positive charge density of the complex, exhibits the characteristics of serum resistance, efficient cell absorption and endosome escape, and low cytotoxicity.
[0024] Compared with the prior art, the application has the following advantages:
[0025] The fluorinated cationic polymer PHP-FH of this invention exhibits superior gene transfection performance as a gene transfection vector. Compared to commonly used PEI gene transfection reagents, it significantly reduces cytotoxicity, achieving highly efficient transfection at extremely low doses and significantly mitigating toxic side effects on cells. Furthermore, the introduced fluorinated alkyl chain endows the polymer with unique amphiphilicity, greatly enhancing the stability of the polymer and genes in serum. Compared to anhydride-fluorinated PHP materials, an inherent advantage of this invention is its superior stability and resistance to degradation. Therefore, using the fluorinated cationic polymer PHP-FH of this invention, not only can highly efficient and low-toxicity transfection of various cell types be achieved in vitro, but it can also be used for highly efficient and low-toxicity local drug delivery in vivo, demonstrating broad potential for clinical and research applications. Attached Figure Description
[0026] Figure 1 Synthetic route diagram for the fluorinated cationic polymer PHP-FH;
[0027] Figure 2 Infrared spectra of PHP and a series of fluorinated PHP;
[0028] Figure 3 NMR fluorine spectra of PHP and series fluorinated PHP;
[0029] Figure 4 Electrophoresis diagram of a gel retardation experiment of a fluorinated PHP / DNA complex;
[0030] Figure 5 Particle size (a) and zeta potential (b) of plasmid DNA, PEI, PHP, and fluorinated PHP complex;
[0031] Figure 6 A schematic diagram illustrating the application of PHP-FH8 as a gene transfection vector in gene transfection;
[0032] Figure 7 Images showing the transfection effect of a series of fluorinated PHP cells on HEK293T cells;
[0033] Figure 8 Images showing the transfection effect of fluorinated PHP on HeLa cells;
[0034] Figure 9 The figures show the results of the MTT assay for the cytotoxicity of a series of fluorinated PHP, where (a) is the result of the HEK293T cytotoxicity test and (b) is the result of the HeLa cytotoxicity test.
[0035] Figure 10The images show the transfection effects of PHP-F6 on various cell types, with (a) showing the transfection effect on HEK293T cells and (b) showing the transfection effect on HeLa cells. Detailed Implementation
[0036] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.
[0037] In the following examples, the preparation of PHP was referenced in [Pan, X.; Cheng, S.; Su, T.; et al., Poly(2-hydroxypropylene imines) functionalized magnetic polydopamine nanoparticles for high-efficiency DNA isolation. Applied Surface Science 2019, 498.].
[0038] Example 1: Synthesis and Characterization of PHP Fluoride
[0039] 1. Synthesis of a series of fluorinated PHP:
[0040] PHP was uniformly dispersed in deionized water to form an aqueous solution. 3-(2,2,3,3-tetrafluoropropoxy)-1,2-propene oxide dissolved in ethanol at 5 times the volume of deionized water was added to the aqueous solution. The mixture was stirred at 25°C for 48 hours. After the reaction was completed, the product was passed through a cellulose membrane with a pore size of 3500 Da. It was first dialyzed in ethanol for 2 days, then dialyzed in water for 2 days, and finally freeze-dried under vacuum to obtain fluorinated PHP, named PHP-FH4.
[0041] Using the above method, 3-(2,2,3,3-tetrafluoropropoxy)-1,2-propene oxide was replaced with 3-(1H,1H,5H octafluoropentoxy)-1,2-propene oxide to prepare fluorinated PHP, which was named PHP-FH8.
[0042] Using the above method, fluorinated PHP was prepared by replacing 3-(2,2,3,3-tetrafluoropropoxy)-1,2-propene oxide with 2-(((3,3,4,4,5,5,6,6,7,7,8,8,8-tetrafluorooctyl)oxy)methyl)ethylene oxide, named PHP-FH13. The synthetic route is shown below. Figure 1 .
[0043] 2. Characterization by infrared spectroscopy and nuclear magnetic resonance fluorine spectrum:
[0044] Infrared spectra of PHP, PHP-FH4, PHP-FH8, and PHP-FH13 are shown below. Figure 2The nuclear magnetic resonance fluorine spectrum is shown in [reference needed]. Figure 3 . Figure 2 The results showed that after fluorination, 1000-1400 cm -1 The vibrational peaks gradually increase within the range due to the introduction of CF bonds, resulting in CF stretching vibrations. This trend is particularly evident in the spectra of PHP-FH8 and PHP-FH13, where the absorption peaks corresponding to CF bonds become increasingly prominent, consistent with the increase in the degree of fluorination. Figure 3 Nuclear magnetic resonance fluorination spectroscopy confirmed the discovery of Fourier transform infrared spectroscopy, confirmed the existence of CF bonds, and allowed for the quantification of fluorination sites, thus confirming the successful fluorination of poly-2-hydroxyacrylamide.
[0045] 3. Determination of the number of fluorinated alkyl grafts:
[0046] The ninhydrin assay was used to determine the number of fluorinated alkyl groups grafted into fluorinated PHP. The specific steps were as follows: First, 0.35 g of ninhydrin was dissolved in 100 mL of ethanol, which could be replaced by an equal volume mixture of isopropanol or butanol and acetone. Next, a 1% protein standard solution was prepared, and different volumes (e.g., 10 μL, 20 μL) of the standard protein solution were transferred to a series of test tubes. Sufficient distilled water was added to each test tube to bring the total volume to 1 mL, thus preparing a standard curve. One blank test tube containing only 1 mL of distilled water was selected, while test tubes 2 to 9 were used to construct the standard curve. Test tubes 10 to 15 were filled with the unknown sample. Next, 1 mL of ninhydrin reagent was mixed with 5 mL of solvent diluent and added to each test tube. After mixing using a vortex mixer, the mixture was heated in a 90°C incubator for 17 minutes or placed in boiling water for 20 minutes. The test tubes were then cooled to room temperature, and the optical density at 570 nm (440 nm for proline or hydroxyproline) was compared for each test tube to construct a standard curve of primary amine concentration. Based on this, the reduction in primary amine in the fluorinated polymer in the unknown sample was calculated, thereby determining the number of fluorinated alkyl grafts.
[0047] The results showed that the grafting rate of fluorinated PHP alkyl chains was between 50% and 65%. Therefore, it can be concluded that fluorinated PHP with a high level of modification can be obtained by modifying PHP with fluorinated compounds of ethylene oxide.
[0048] Comparative Example 1: Preparation of the fluorinated cationic polymer PHP-F6
[0049] Synthesize PHP-F6 according to the following reaction route:
[0050]
[0051] The specific steps are as follows: PHP is evenly dispersed in deionized water to form a PHP aqueous solution. Ethylene oxide (2,2,3,3,4,4,4-heptafluorobutyl) dissolved in ethanol at 5 times the volume of deionized water is added to the PHP aqueous solution. The mixture is stirred and reacted at 25°C for 48 hours. After the reaction is completed, the product is passed through a cellulose membrane with a pore size of 3500 Da. It is first dialyzed in ethanol for 2 days, then dialyzed in water for 2 days, and then freeze-dried under vacuum to obtain fluorinated PHP, named PHP-F6.
[0052] Example 2: Preparation and characterization of fluorinated PHP / DNA complex
[0053] 1. Preparation of fluorinated PHP / DNA complex:
[0054] Fluorinated cationic polymers PHP-FH4, PHP-FH8, and PHP-FH13 were dissolved in PBS buffer (pH 7.2–7.4) to form fluorinated PHP solutions, while plasmid DNA was dissolved in deionized water to form plasmid DNA solutions. Using 1 μg of plasmid DNA, the mass ratios of fluorinated PHP to plasmid DNA were 0.5:1, 1:1, 2:1, 4:1, and 8:1, respectively. The fluorinated PHP solutions and plasmid DNA solutions were thoroughly mixed and uniformly pipetted, then allowed to stand at room temperature for 30 minutes to obtain the fluorinated PHP / DNA complex solution.
[0055] 2. Gel retardation experiment:
[0056] 20 μL samples were taken from the above-mentioned fluorinated PHP / DNA complex solution and subjected to agarose gel electrophoresis to assess DNA retardation. Electrophoresis was performed on a 0.8% agarose gel using electrophoresis buffer containing GoldViewer™, at a voltage of 90 V, and for 60 minutes.
[0057] Electrophoresis results as follows Figure 4 As shown, this effectively demonstrates one of the core requirements for gene delivery: compressing nucleic acids into nanoparticles to prevent them from being damaged before entering cells. Figure 4 The study showed that when the mass ratio reached 1:1, all migration streaks of plasmid DNA completely disappeared, proving that even at a lower ratio, fluorinated PHP can effectively compress and protect DNA.
[0058] 3. Physicochemical characterization of fluorinated PHP / DNA complex
[0059] The particle size and zeta potential of the above-mentioned fluorinated PHP / DNA complex were determined using a nanoparticle size analyzer, and the results are as follows: Figure 5 As shown.
[0060] Figure 5The particle size and zeta potential of the composites at different mass ratios were revealed. Figure 5 As shown in Figure (a), as the mass ratio increases from 0.5 to 2, the average particle size of the complex decreases significantly from approximately 1400 nm to approximately 300 nm. This change indicates that as the ratio of carrier to DNA increases, the size of the complex tends to be smaller, which is more suitable for cellular uptake and helps prevent DNA from being degraded by enzymes in the extracellular environment. Figure 5 Figure (b) shows the zeta potential of the complex at different mass ratios. The zeta potential of the initial DNA sample was approximately -30 mV, while as the mass ratio increased, the zeta potential of the complex gradually increased, approaching neutral or slightly positive charge, which facilitates the complex's passage through the negatively charged cell membrane.
[0061] Furthermore, a low to moderate cation charge density is beneficial for reducing cytotoxicity because it avoids cell membrane damage that may be caused by excessively high cation charge densities. Based on the above measurements, it can be concluded that the physicochemical properties of the fluorinated PHP / DNA complex fully meet the expected requirements for efficient and low-toxicity gene transfection.
[0062] Example 3: Samples were stored at 4°C for six months, and cell transfection experiments using fluorinated PHP / DNA complex were conducted.
[0063] Fluorinated cationic polymers PHP-FH4, PHP-FH8, and PHP-FH13, stored at 4°C for six months, were used to transfect plasmid DNA containing green fluorescent protein (GFP). HEK293T and HeLa cells were targeted. First, the fluorinated cationic polymers PHP-FH4, PHP-FH8, and PHP-FH13 were dissolved and sterilized under ultraviolet light, then mixed with plasmid DNA at an optimal 1:1 mass ratio. Cells were seeded in 24-well plates at a cell density of 1.0 × 10⁶ cells per well. 5 Cells were cultured for 18–24 hours to achieve approximately 70% cell confluence. Before transfection, cells were washed with PBS buffer, and then 300 μL of serum-free medium containing the complex (100 μL of the complex PBS solution and 200 μL of serum-free medium) was added to each well, and incubated at 37°C for 4 hours. After incubation, the medium was replaced with fresh medium containing 10% serum, and cultured for another 24 hours. GFP expression in the cells was examined using confocal microscopy. Transfection results are shown below. Figure 7 and Figure 8 As shown, Figure 7 The transfection effect of HEK293T cells was demonstrated, and Figure 8 This shows the transfection status of HeLa cells. (From...) Figure 7 and 8 It is evident that fluorinated PHP exhibits excellent transfection performance.
[0064] Example 4: MTT assay for the cytotoxicity of fluorinated PHP
[0065] HEK293T cells and HeLa cells in logarithmic growth phase were digested with 0.02% EDTA + 0.25% trypsin to prepare cell suspensions, and then subjected to 1×10⁻⁶ ppm of each suspension. 4 Cells were added to a concentration of 200 μL / mL in a 96-well ELISA plate, with five replicates per well. The plates were incubated at 37°C in a 5% CO2 incubator for approximately 24 hours. The medium was replaced with 200 μL of fresh medium, and then a complex of fluorinated PHP and plasmid DNA was added to each well (N / P ratios of 0, 1:1, and 2:1), and incubated for 24 hours. Then, 20 μL of LTT solution (in 5 mg / mL PBS) was added to each well, and the plates were incubated for another 4 hours. All supernatant was discarded, and 100 μL of DMSO was added to each well. The plates were shaken for 10 minutes on a microplate reader, and the absorbance was measured at 570 nm. Higher absorbance values indicate a higher number of viable cells.
[0066] Wherein, cell viability = OD 样品 / OD control ×100%. Where OD 样品 The absorbance value at 570 nm for cell groups treated with fluorinated PHP / DNA complex solution is OD. control The absorbance value at 570 nm is for cell groups treated with culture medium only.
[0067] In this embodiment, PHP and PEI 25kDa were used as negative and positive controls, respectively. The evaluation was performed directly by comparing PHP and PEI 25kDa materials with the same mass ratio. Figure 9 As shown, in the 4-hour cytotoxicity test, the cell viability of the complexes transfected with PHP and fluorinated PHP at various proportions was >50%, and the cell viability of cells transfected with fluorinated PHP was still significantly higher than that of cells transfected with PHP. It can be seen that the fluorinated PHP of this invention has significantly better performance than PEI 25kDa, and the advantages of fluorinated PHP are obvious at high concentrations, with significantly reduced cytotoxicity and extremely high biocompatibility.
[0068] Comparative Example 2: Cell Transfection Assay of PHP-F6 / DNA Complex
[0069] Transfection experiments using PHP-F6 and plasmid DNA containing green fluorescent protein (GFP) were conducted on HEK293T and HeLa cells. First, PHP-F6 was dissolved and sterilized with UV light, then mixed with plasmid DNA at a 1:1 optimal mass ratio. Cells were seeded in 24-well plates at a cell density of 1.0 × 10⁶ cells per well. 5Cells were cultured for 18–24 hours to achieve approximately 70% cell confluence. Before transfection, cells were washed with PBS buffer, and then 300 μL of serum-free medium containing the complex (100 μL of the complex PBS solution and 200 μL of serum-free medium) was added to each well, and incubated at 37°C for 4 hours. After incubation, the medium was replaced with fresh medium containing 10% serum, and cultured for another 24 hours. GFP expression in the cells was examined using confocal microscopy. Figure 10 This diagram illustrates the application of PHP-F6 as a gene transfection vector in gene transfection. Figure 10 (a) shows the transfection effect of HEK293T cells, while Figure 10 (b) shows the transfection status of HeLa cells. It is evident that the transfection effect of PHP-F6 is far inferior to that of the materials in the examples.
[0070] The fluorinated cationic polymer gene vector PHP-FH prepared in this invention exhibits a large number of hydroxyl and fluorinated alkyl chains, as revealed by NMR spectroscopy. Ninhydrin analysis showed that the number of grafted fluorinated alkyl chains on a single polymer molecule could reach up to 41. Dynamic light scattering (DLS) analysis of the complex particle size demonstrated that the fluorinated cationic polymer of this invention has a stronger DNA-binding capacity, and the formed complex exhibits excellent stability. Electrophoretic light scattering (ELS) analysis of the zeta potential indicated that the moderate positive charge of the complex also significantly reduced the polymer's cytotoxicity. Using the fluorinated polymer material of this invention as a gene vector for transfecting human embryonic kidney cells HEK293T and human cervical cancer cells HeLa, the transfection efficiency was 12%–25% higher than that of commercially available PEI 25kDa. After six months of storage, the material still maintained stable transfection efficacy. MTT cytotoxicity testing showed a significant reduction in cytotoxicity. Compared with commercially available PEI 25kDa, cell viability increased by 10%–20% at a low N / P ratio, and the increase was even greater at a high N / P ratio, with cell viability increasing by over 30% at N / P = 8. The vector prepared using this invention can achieve highly efficient and low-toxicity transfection of various cells in vitro, and can be used for highly efficient and low-toxicity local drug delivery in vivo, showing broad application prospects.
[0071] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the present invention, and all such modifications and improvements fall within the scope of the present invention.
Claims
1. Fluorinated cationic polymer, characterized in that, The structural formula is as follows: ， In the formula, x is an integer greater than 0, y is an integer greater than or equal to 0; R is H or a polyfluoroalkyl chain, and part of R is not H, and the polyfluoroalkyl chain is selected from one of the following: 。 2. The fluorinated cationic polymer according to claim 1, characterized in that, The molecular weight is 3500-13000.
3. The method for preparing a cationic fluorinated polymer according to claim 1 or 2, characterized in that, The specific steps are as follows: An ethylene oxide fluorine-containing compound is added to the PHP solution, and the reaction is stirred at room temperature. After the reaction is completed, the product is dialyzed in ethanol and water in sequence, vacuum freeze-dried, and a fluorinated cationic polymer PHP-FH is obtained, and the ethylene oxide fluorine-containing compound is selected from one of the following: 。 4. The production method according to claim 3, characterized by, The molar ratio of PHP to the ethylene oxide fluorine-containing compound is 1: (30-100).
5. The preparation method according to claim 3, characterized in that, The stirring reaction time is 2-3 days.
6. The preparation method according to claim 3, characterized in that, The specific method of dialysis is: through a cellulose membrane with a pore size of 3500 Da or more, first dialyzed in ethanol for 2-4 days, and then dialyzed in water for 2-4 days.
7. The preparation method according to claim 3, characterized in that, The vacuum freeze-drying method is: first pre-freeze at -80℃ for 1 day, and then vacuum freeze-dry for 2-4 days.
8. The fluorinated cationic polymer according to claim 1 or 2 as a gene transfection carrier for gene transfection.
9. Use according to claim 8, characterized in that, The mass ratio of the fluorinated cationic polymer to the loaded gene is (1-50):
1.
10. Use according to claim 8, characterized in that, The gene is DNA, siRNA, shRNA, microRNA, or modified nucleic acid.