1. Introduction
Gene therapy is a promising therapeutic method for delivering nucleic acid agents or genome-editing tools into diseased cells [
1]. Initial research focused on the development of viral vectors including adenoviruses and retroviruses which have high efficiency toward delivering nucleic acids [
2]. However, what troubles us is the toxicity, immunogenicity and limitations of scaling up these procedures [
3]. Thus, one of the great challenges of gene delivery is to design novel non-viral vectors that fulfill the promise of high delivery efficiency and high safety [
4]. Various non-viral systems, such as cationic liposomes [
5], polymers [
6], inorganic nanoparticles [
7] and quantum dots [
8], have been developed to improve the gene delivery properties. Among these types, cationic polymers have attracted great attention because of some remarkable advantages including facile manufacturing, high nucleic acid capacity, good stability and easily modification [
7].
Polyethylenimine (PEI) is the most studied cationic polymer for gene delivery, and branched PEI 25 kDa has been considered as the golden standard for new polymeric non-viral vectors on account of its high transfection efficiency (TE), which is attributed to the strong buffering capacity in the pH range of 7.4–5.1 [
9]. Although high molecular weight PEI shows good TE, it also exhibits obvious cytotoxicity for its non-degradable and highly positively charged structure [
10,
11]. Reducing molecular weight could solve the toxicity problem, but the TE is sacrificed [
12]. To reduce surface charge and prolong the circulation time of cationic nanoparticles, polyethylene glycol (PEG) or other hydrophilic chains have been introduced to high molecular weight PEI [
13]. On the other hand, low molecular weight (LMW) PEI could also be connected by degradable linkers including ester [
14], disulfide [
15] and β-aminoester [
16] to achieve enhanced TE and reduced toxicity. Beside PEI, another kind of classical polycation is poly-
L-lysine (PLL) [
17], which can get rid of biological toxicity and system side effects for its polypeptide chain connected by amide bond that can be degraded by an internal enzyme [
18]. However, since all amine groups in PLL are protonated primary amines [
19], PLL has no pH buffering capacity at physiological environment. Meanwhile, histidine can be cited as a modification group in non-viral gene vectors for its good pH buffering capacity from the imidazole ring, especially in the more acidic endosomes and lysosomes [
20].
Recently, we developed some polycations via Michael addition between LMW PEI 600 Da and diacryl esters, and these materials exhibited promise as non-viral gene vectors with higher TE and lower toxicity compared to PEI 25 kDa [
21]. Herein, we combine the advantages of cationic PEI and amino acids with their special functions. The designed polycations were constructed from PEI 600 Da and linkages modified by amino acids such as lysine and histidine. Lysine can strengthen the DNA binding ability of the vector, while histidine has the potential to improve the pH buffering capacity. The low molecular weight of PEI and the proteinogenic common amino acids gave the polymers much lower cytotoxicity. Results demonstrated that the title vectors were able to condense DNA into nanoparticles with good stability. Furthermore, these materials showed higher TE and lower toxicity comparing to PEI 25 kDa whether with the presence of serum or not. The results reveal that such cationic polymers could serve as promising candidates for non-viral gene delivery.
3. Experimental Section
3.1. Materials and Methods
All chemicals used in experiments were bought from commercial sources and used without additional purification unless otherwise stated. Dichloromethane and methanol were dried with proper desiccants and distilled immediately before use. Column chromatography was performed by 200–300 mesh silica gel or Al2O3. Deionized water was used to prepare all aqueous solutions. Branched polyethylenimine (bPEI 25 kDa) was purchased from Sigma-Aldrich. The plasmids used in this study re pEGFP-N1 (Clontech, Palo Alto, CA, USA) coding for EGFP DNA and pGL-3 (Promega, Madison, WI) coding for luciferase DNA. The Micro BCA protein assay kit was obtained from Pierce (Rockford, IL, USA). Cy5 was bought from Mirus Bio, LLC (Madison, WI, USA). The luciferase assay kit and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H tetrazolium, inner salt) were purchased from Promega (Madison, WI, USA). Endotoxin-free plasmid purification kit was bought from TIANGEN (Beijing, China). The 1H NMR and 13C-NMR spectra were measured on a Bruker AM400 NMR spectrometer. Proton Chemical shifts of NMR spectra were given in ppm relative to internals reference TMS (1H, 0.00 ppm). HRMS (ESI) spectral data was measured on a Bruker Daltonics Bio TOF mass spectrometer. We used a gel permeation chromatography system (GPC) to measure the molecular weights (Mw) and the polydispersity index (PDI, Mw/Mn) of the polycations. The GPC consisted of a Waters 515 pump, a linear 7.8 × 300 mm column (Waters Corp, Milford, MA, USA), an 18 angle laser scattering instrument (Wyatt Technology Corporation, Santa Barbara, CA, USA), and an OPTILAB DSP interferometric refractometer (Wyatt Technology Corporation, Santa Barbara, CA, USA). 0.5 M NaOAc/0.5 M HOAc (pH 4.6) passed through a 0.02 μm film filter was used as the eluent. The flow rate was 1 mL min−1.
3.2. Synthesis and Characterization of Compound B
2Boc-Lys was prepared according to the method reported before21. 2Boc-Lys (5 g, 14.4 m mol) was dissolved in dichloromethane (DCM), then N-methylmorpholine (1.75 g, 17.3 mmol) and isobutyl chloroformate (2.37 g, 17.3 m mol) was add to the solution at 0 °C. After stirring for 0.5 h, diethanolamine (1.82 g, 17.3 m mol) was add to the solution and stirred overnight at room temperature. When the reaction completed, the reaction mixture was washed with saturated aqueous NaHCO3 solution, water and brine in sequence. After dried with anhydrous Na2SO4, the organic layer was distilled under reduced pressure and the residue was purified by silica gel column chromatography (v/v 10:1, DCM-CH3OH) to give compound A as colorless or yellowish thick oil. Yield: 40%. 1H-NMR (400 MHz, CDCl3, TMS): δ = 1.41, (s, 18H, -Boc), 1.47‒1.88, (m, 4H, -CH2CH2CH2NHBoc-), 3.06, (m, 2H, -CH2CH2CH2CH2NHBoc), 3.46, (m, 2H, -CH2NHBoc), 3.60–3.66, (m, 4H, -CONCH2CH2O-), 3.82, (m, 4H, -CONCH2CH2O-), 4.22, (m, 1H, -CH-), 4.59‒4.71, (m, 2H, -OH). 13C-NMR (CDCl3, 100 MHz): δ = 22.3, 28.3, 29.5, 32.3, 39.9, 50.3, 51.9, 60.4, 80.1, 156.1, 174.4. HR-MS (ESI): Calcd for: C20H39N3O7: 456.2686 [M + Na]+; Found: 456.2686 [M + Na]+.
Compound A (2 g, 4.61 m mol) and triethylamine (1.12 g, 11.07 m mol) were dissolved in anhydrous DCM. Acryloyl chloride was dissolved in dry DCM and then added dropwise to the stirred solution in the ice bath. The mixture was stirred overnight at room temperature. After that, the mixture was washed by saturated aqueous NaHCO3 solution, water and brine in sequence, followed by evaporation of the volatile solvent. The residue was purified with silica gel column chromatography (v/v 1:1, PE-EA) to give compound B as colorless thick oil. Yield: 59%. 1H-NMR (400 MHz, CDCl3, TMS): δ = 1.43 (s, 18H, -Boc), 1.25‒1.31 (m, 2H, -NCONHCHCH2CH2-), 1.49‒1.57 (m, 2H, -CH2CH2NH-Boc), 1.62 (m, 2H, -NCONHCHCH2-), 3.09 (m, 2H, -CH2NH-Boc), 3.42‒3.92 (m, 4H, -OCH2CH2N-), 4.26‒4.39 (m, 4H, -OCH2CH2N-), 4.66 (m, 1H, -CH-), 5.83‒5.88 (m, 2H, -OCCHCH2), 6.07‒6.18 (m, 2H, -OCCHCH2), 6.38‒6.44 (m, 2H, -OCCHCH2). 13C-NMR (100 MHz, CDCl3): δ = 22.5, 28.4, 33.4, 40.2, 45.6, 47.1, 49.9, 61.8, 79.7, 127.9, 131.5, 156.0, 165.0, 173.1. (HR-MS (ESI): Calcd for: C20H39N3O7: 564.2892 [M + Na]+; Found: 564.2917 [M + Na]+.
3.3. Synthesis and Characterization of Compound D
Firstly, 2Boc-His and compound
C were prepared according to the method reported before [
21,
26]. Then, the protecting t-butyloxycarbonyl (Boc) group of compound
C (5 g, 16.12 m mol) was removed by trifluoroacetic acid (TFA) at 0 °C in the DCM solution. After removing the solvent and most TFA, the residues were dissolved in 50 mL of DCM. In total, 5 mL of NH
3·H
2O was dissolved in 50 mL of water and added to the DCM solution until the pH of organic phase reached ˃7. The organic phase was separated from mixture and dried with anhydrous Na
2SO
4. Then, 2Boc-His (4.60 g, 13.29 m mol), 1-hydroxybenzotriazole (2.03 g, 13.29 m mol) and
N,
N-diisopropylethylamine (1.72 g, 13.29 m mol) were added to the organic phase. 1-Ethyl-3-(3-dimethyl propyl) carbodiimide was dissolved in dry DCM and then added dropwise to the stirred solution at 0 °C. After stirring overnight, the mixture was washed by saturated NaHCO
3 solution, water and brine in sequence, followed by evaporation of the volatile solvent. The residue was purified with silica gel column chromatography (
v/v 1:1, PE-EA) to give compound
D as white or yellowish powder. Yield: 13.0%.
1H-NMR (400 MHz, CDCl
3, TMS): δ = 1,39 (s, 9H, -CHNHBoc), 1.59 (s, 9H, -NBoc), 2.83–2.91 (m, 4H, -NH
2CHC
H2-), 3.45‒3.85 (m, 4H, -OCH
2C
H2N-), 4.28 (m, 4H, -OC
H2CH
2N-), 4.96‒4.98 (m, 1H, -C
HNH-), 5.79‒5.86 (m, 2H,-OCC
HCH
2), 6.06‒6.16 (m, 2H, -OCCHC
H2), 6.37‒6.41 (m, 2H, -OCCHC
H2), 7.15 (s, 1H, -C
HNBoc), 7.96 (s, 1H, -NC
HNBoc).
13C-NMR (100 MHz, CDCl
3): δ = 27.9, 32.5, 47.3, 49.8, 62.1, 79.7, 85.4, 114.7, 128.0, 131.4, 136.8, 138.6, 146.9, 155.0, 165.8, 172.4. HR-MS (ESI): Calcd for: C
20H
39N
3O
7: 573.25331 [M + H]
+; Found: 573.2566 [M + H]
+.
3.4. Synthesis and Characterization of Target Polymers LysP and HisP
Briefly, bPEI 600 (300 mg, 0.5 m mol) and compound B or D (0.5 m mol) were separately dissolved in 1 mL of anhydrous methanol and 1 mL of anhydrous DCM. The reaction mixtures were refluxed at 45 °C with constant stirring for 72 h. After that, the mixtures were diluted with 20 mL of DCM, and 5 mL of TFA was added with stirring overnight to remove the protecting Boc group of polymers. After removing the solvent and most TFA, the residues were dissolved in 1 mL of ethanol and precipitated by the addition of diethyl ether. The precipitation was collected and dried in vacuum to get the product as yellow viscous solid. The molecular weights of LysP and HisP were measured by GPC.
LysP. 58.5% yield. Mw: 9115 Da, PDI: 1.97. 1H-NMR (400 MHz, D2O, TMS): δ = 1.27‒1.29, (m, 2H, -NCONHCHCH2CH2-), 1.50‒1.52, (m, 2H, -CCH2CH2CH2CH2NH2), 1.72‒1.74, (s, 2H, -NCONHCHCH2-), 2.55‒3.36, (m, 54H, -NHCH2CH2N- and - CCH2CH2CH2CH2NH2),
HisP. 52.7% yield. Mw: 7208 Da, PDI: 1.79. 1H-NMR (400 MHz, DMSO, TMS): δ = 2.48–3.47 (m, 60H, -NHCH2CH2N- and -NH2CHCH2C-), 7.40 (s, 1H, -NHCHC-), 9.0, (m, 1H, -NHCHN-).
3.5. Agarose Gel Retardation Assay
LysP/DNA and HisP/DNA complexes at different weight ratios ranging from 0.5 to 3.2 were prepared by adding an appropriate volume of LysP/HisP to 5 μL of pUC-19 (0.025 mg/mL). The obtained complex solutions were diluted to 10 μL, and then all the complexes were incubated at 37 °C for 30 min. Then, the complexes were electrophoresed on a 1% (w/v) agarose gel containing GelRed™ and with Triseacetate (TAE) running buffer at 140 V for 30 min. DNA was visualized under an ultraviolet lamp using a Vilber Lourmat imaging system.
3.6. Ethidium Bromide Displacement Assay
The ability of LysP and HisP to condense DNA was studied by using EB exclusion assays. Fluorescence spectra were measured at room temperature in air by a Horiba Jobin Yvon Fluoromax-4 spectrofluorometer and corrected for the system response. EB (2.5 mL, 1 mg/mL) was added into quartz cuvette containing 2.5 mL of 10 mM Hepes solution. The fluorescence intensity of EB was measured after enough shaking. Then CT DNA (10 mL, 1 mg/mL) was added to the solution and mixed symmetrically, and the measured fluorescence intensity was the result of the interaction between DNA and EB. Subsequently, the solution of polymers (1 mg/mL, 2 mL for each addition) was added to the above solution for further measurement. All the samples were excited at 520 nm and the emission was measured at 600 nm. The pure EB solution and DNA/EB solution without cationic polymer were respectively used as negative and positive controls. The percent relative fluorescence (%F) was determined using the equation %F = (F−FEB) / (F0−FEB), wherein FEB and F0 denote the fluorescence intensities of pure EB solution and DNA/EB solution, respectively.
3.7. Particle Size and ζ-Potential Measurement in Water
Zeta potential (ζ-potential) and size of the polyplex particles were measured by Nano-ZS ZEN3600 apparatus (Malvern Instruments) at 25 °C. The complexes with various weight ratio of polycations were prepared by adding 1 mg of pUC-19 to appropriate volume of the deionized water. Before measurement, the solution of the complexes was incubated at 37 °C for 0.5 h and then diluted with deionized water to 1 mL. The data were shown as mean ± standard deviation (SD) based on triplicate independent measurement.
3.8. Transmission Electron Microscopy (TEM)
The morphologies of the polyplexes were observed by TEM (JEM-100CXa) with an acceleration voltage of 100 kV. 1 mg of pUC-19 was added to the appropriate volume of the polymer solution (optimal weight ratio of each sample), then diluted to the total volume of 50 μL. The solution of the polyplexes was incubated at 37 °C for 0.5 h. The polyplex solution was diluted with deionized water to 1 mL before measurement. A drop of DNA/polymer complexes solution was placed onto the copper grid. The excess solution was blotted away with filter paper after a few minutes. Then, a drop of 0.5% (w/v) phosphotungstic acid was placed on the above grid. The grid was dried at room temperature for several minutes before observation.
3.9. Cell Culture
HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin-streptomycin, 10 KU mL−1) at 37 °C in a humidied atmosphere containing 5% CO2. B16 and 7702 cells were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640) with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin-streptomycin, 10 KU mL−1) at 37 °C in a humidied atmosphere containing 5% CO2.
3.10. Amplification and Purification of Plasmid DNA
pGL-3 plasmids were transformed in M109 Escherichia coli and seed as the luciferase reporter gene. pEGFP-N1 plasmids is the enhanced green fluorescent protein reporter gene which was transformed in E. coli DH5a. Both plasmids were amplified in E. coli grown in LB medium at 37 °C and 220 rpm overnight. The plasmids were purified by an EndoFree TiangenTM Plasmid Kit. Then, the purified plasmids were dissolved in TE (Tris + EDTA) buffer solution and stored at −80 °C. The integrity of plasmids was confirmed by agarose gel electrophoresis. The purity and concentration of plasmids were determined by the ratio of ultraviolet (UV) absorbances at 260–280 nm.
3.11. Gene Transfection Assay
Gene transfection of a series of complexes was investigated in HeLa, B16 and 7702 cells. Cells were seeded in 48-well plates (5 × 104 cells/well) and grown to 70%–80 % cell confluence at 37 °C for 24 h in 5 % CO2. Before transfection, the medium was exchange into a serum-free or a serum-containing culture medium containing polymer/pDNA (0.4 μg) complex at various weight ratios. The medium was replaced with fresh medium containing serum and incubated for another 20 h after 4 h standard incubator conditions.
For fluorescent microscopy assays, cells were transfected by complexes containing pEGFP-N1. After 24 h incubation, the cells expressed pEGFP-N1 were observed with an inverted fluorescence microscope (Nikon Eclipse TE 2000E, Tokyo, Japan) equipped with a cold Nikon camera. Control transfection was performed in each case using a commercially available transfection reagent bPEI 25 kDa based on the standard conditions specified by the manufacture.
For luciferase assays, cells were transfected with complexes containing pGL-3 plasmids. After 24 h transfection as described before, the luciferase assay was performed according to the manufacturer’s protocols (Promega). The luciferase activity was measured by microplate reader (Model 550, BioRad, Hercules, CA, USA). The protein content of the lysed cell was determined by BCA protein assay kit (Pierce). Gene transfection efficiency was expressed as the relative fluorescence intensity per mg protein (RLU/mg protein). bPEI 25 kDa and bPEI 600 Da were used as control. All experiments were performed in triplicate.
3.12. Cell Viability Assay
Toxicity of LysP and HisP toward HeLa cells, B16 cells and 7702 cells was determined by an MTS reduction assay. The cells mentioned above were seeded 1 × 104 cells/well into 96-well plates for 24 h at 37 °C in a humidied atmosphere containing 5% CO2. The medium was replaced with 100 mL of fresh medium without FBS, to which 100 mL complexes at various weight ratio of polymer relative to pDNA was added to achieve final volume of 200 mL. The cells were then incubated in the medium without FBS containing polymer/pDNA (0.2 mg) complexes at various weight ratios. The polyplexes solutions were removed after 24 h of incubation. 20 mL of MTS and 80 mL of PBS were added to each well for extra 1 h incubation. In the measurement of B16 cells, RPMI-1640 medium was used instead of PBS. The absorbance was measured by a microtiter plate reader. The cell viability (%) was obtained according to the manufacturer’s instructions as follows: cell viability = (ODtreated/ODcontrol) × 100%. The bPEI 25 kDa and bPEI 600 Da were used as control. All experiments were performed in triplicate.
3.13. Cellular Uptake of Plasmid DNA
The cellular uptake of the polymer/Cy5-labeled DNA complexes was analyzed by flow cytometry. The Label IT Cy5 Labeling Kit was used to label pDNA with Cy5 according to the manufacturer’s protocol. The cells mentioned were cultured 1 × 105 cells/well in 24-well plates for 24 h at 37 °C in a humidied atmosphere containing 5% CO2 before in vitro gene transfection. Then, the B16 cells were incubated with the Cy5-labeled polyplexes (0.8 μg DNA/well, optimal weight ratio of each sample) for 4 h at 37 °C in different serum-containing medium and uptake efficacy was analyzed using flow cytometry. After that, the cells were washed with 1 × PBS and harvested with 0.25% Trypsin/EDTA and resuspended in 1 × RPMI-1640. The uptake of Cy5-labeled plasmid DNA was measured in the FL4 channel using the red diode laser (633 nm). Mean fluorescence intensity was analyzed using flow cytometer (Becton Dickinson and Company, Franklin Lakes, NJ, USA).
3.14. Confocal Laser Scanning Microscopy (CLSM) Analysis
To investigate the cellular uptake of the complexes, Cy5-labeled DNA was used to monitor intracelluar trafficking behaviors (0.8 μg DNA/well, optimal weight ratio of each sample). The B16 cells were incubated with the polyplexes for 4h. The nuclei of B16 cells were stained with hoechst 33342. Co-localizations of the polyplexes with acidic vesicles in B16 cells were observed by the CLSM observation which was performed using LSM 780 (Zeiss) at excitation wavelengths of 405 nm for hoechst 33342 (blue), 633 nm for Cy5 (red).
3.15. Biodegradation of Polymers
LysP and HisP was dissolved in 1 × PBS buffering solution and incubated in the shaker incubator at 37 °C, 100 rpm. The polymer was sampled at different time points and lyophilized. The Mw and PDI was measured by GPC.