b. Department of Plastic Surgery, First A liated Hospital of Anhui Medical University, Hefei 230032, China
Nowadays, gene therapy can be considered as an efficient and minimally invasive medical treatment method, especially for diseases caused by genetic defects [1-9]. Since it was presented in 1967, various techniques have been extensively studied to introduce foreign DNA into mammalian cells [10-15]. The gene delivery by using a viral or non-viral gene carrier currently seems to be one of the best methods for gene transportation [16, 17], although there are many challenges during the gene transfer process, such as serum aggregation, unspecific cellular uptake, and degradation by endogenous enzymes [18, 19]. Compared with viral carriers, non-viral carriers are more hopeful due to the biosafety reason [20, 21]. Cationic lipsome and polyethylenimine (PEI) have been tried to replace the viral carriers . However, their cytotoxicity and poor biocompatibility still produce a risk to throw patients into a new danger [23, 24].
Chitosan (CS), which is obtained by the deacetylation of the naturally-existing chitin, has been reported to be non-toxic and biocompatible both in animals and humans, and biodegradable in vivo [25-28]. CS can be positively charged in acidic condition, and apt to coacervate with negatively charged plasmid. The formed CS/plasmid complex particles can availably penetrate the cell membrance , which means CS can carry plasmid easily and safely to the cytoplasm, and be degraded naturally after the gene therapy . However, scientists have long been struggling to solve the problem that the gene transfection efficiency of CS-based vectors is rather low compared to other vectors with relatively high toxicity [31-38]. The molecular weight (MW) of CS is one of the important factors affecting the gene transfection efficiency of CS/plasmid complex. The CS produced from chitin generally has a MW of several 106 kDa. The high MW makes CS hard to be dissolved in water at neutral pH and brings much chain entanglemant, resulting in the dissociation problems, slow degradation in vivo, and finally, the risk of accumulation in the tissues over long period of administration [39-41]. Köping-Höggård et al. reported that CS oligomer (MW < 5 kDa) has higher transfection efficiency than CS with high MW owing to the better ability to release plasmid DNA from the complex particles . Buschmann et al. also reported the degradation of chitosan through nitrous acid method . Based on the investigations on the influence of pH, degree of deacetylation, serum, and other factors on the transfection efficiency of the degraded CS, they declared the better performance of CS with low MW in gene transfection [43, 44]. However, the chemical degradation process of CS involves complex chemical agents and conditions [45, 46] so that the obtained low-MW CS needs to be separated and purifed to ensure its biosafety. γ-Ray can be used to scission CS molecular chains to get high-purity low-MW CS, which is easy to operate and has little influence on the structure of CS [47, 48]. The γ-ray-radiation-scissioned CS has been extensively studied to develop antioxidants, antimicrobials, and absorbents [49-51], but few report on the application as gene vectors. On the other hand, high-MW CS can provide better extracellular DNA protection effect than low-MW CS . Thus, an optimum MW should be achieved for CS in order to obtain high levels of gene transfection.
In this work, the commercial CS was firstly irradiated by γ-ray in an acetic acid solution. Then pEGFP was directly added into the irradiated solution, and coacervated with the γ-ray-radiation-scissioned CS to form pEGFP/CS complex particles. The MW and cell toxicity of CS irradiated at different absorbed dose were investigated. The in vitro gene transfection efficiency of the pEGFP/γ-ray-radiation-scissioned CS complex particles was also investigated by fluorescence microscope and flow cytometry.Ⅱ. MATERIALS AND METHODS A. Reagents and Materials
Analytical reagents including acetic acid, ethanol, NaOH, and sodium sulfate were obtained from Sinopharm Chemical Reagent Co., Ltd. Chitosan (Mw≈3.0×10-5, 91% deacetylation, biological reagent) and ethylene diamine tetraacetic acid (EDTA) were provided by Aladdin Chemistry Co., Ltd. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, >98%) was supplied by Beyotime Institute of Biotechnology. Agarose, ethidium bromide (EB), and polyethylenimine (PEI, branched, ~10-4 Da) were purchased from Sigma-Aldrich Co., LLC. Deionized water was used in all the experiments.
Enhanced green fluorescent protein plasmid (pEGFP) and human cervix epithelial (Hela) cells were provided by the Laboratory of Immunology, Anhui Medical University, Hefei, China. Hela cells were cultured in complete Dulbecco's Modified Eagle's Medium (DMEM, Hyclone), which contained 10% fetal bovine serum (FBS) (Invitrogen, USA) and 1% penicillin-streptomycin inside (Invitrogen, USA), at 37 ℃ under an air atmosphere containing 5%CO2.B. Preparation of γ-ray-radiation-scissioned chitosan
First, 10 mg of CS powder was dissolved in 10 mL of 0.1 mol/L acetic acid aqueous solution. After being purged with nitrogen for 10 min, the system was then irradiated by 60Co γ-ray (2×1014 Bq, located in University of Science and Technology of China) at a dose rate of 83 Gy/min and different total absorbed dose. The irradiated CS sample was denoted as CS-X, X is the value of the total absorbed dose with a unit of kGy.C. Preparation of pEGFP/CS-X complex particles
pEGFP/CS-X complex particles were prepared using complex coacervation method [29, 37]. Firstly, the pH of the irradiated acetic acid solution of CS-X at a concentration of 1 mg/mL was adjusted to 5.5 with the NaOH solution (0.25 mol/L). pEGFP was dissolved in sodium sulfate solution (50 mmol/L) at a concentration of 76 μg/mL. Then, the irradiated CS-X solution and pEGFP solution were vortex mixed at a certain volume ratio for 30 s. After that, the total volume of the mixture was diluted to 100 μL with DMEM, and stood for 30 min. The N/P ratio (the mole ratio of the amine groups in CS-based materials to those of the phosphate ones in pEGFP) in the mixture was set as 20 according to the following equation:
where c and V stand for the concentration and the volume of the CS solution respectively. DD is the deacetylation degree of CS. The number of 205 and 163 are the molecular weights of the structure unit of chitin and deacetylated chitin respectively. mpEGFP is the weight of pEGFP. The number of 660 is the average MW of a base pair of double-stranded DNA .D. Characterizations
FT-IR spectra of irradiated CS were measured on a Bruker VECTOR-22 IR spectrometer using KBr pellets in the range from 4000 cm-1 to 400 cm-1.
The element analysis of the irradiated CS was conducted on a Thermo ESCALAB 250.
The MW of the irradiated CS was measured by viscometric method using Ubbelohde viscometer at 25±0.1 ℃ . The viscosity-average molecular weight of irradiated CS is calculated by the following equation:
where, [η] is the intrinsic viscosity, obtained by the extrapolation mapping of ηsp/c to c and lnηr/c to c. ηsp is the specific viscosity, ηr is the relative viscosity, c is the concentration of the solution. K=1.8×10-3 cm3/g and α=0.93, for CS .
The sizes and zeta potentials of pEGFP/CS-X complex particles were measured on Zetasizer Nano (NANO ZS90, Malvern Inst. Ltd. Malvern, UK).E. Cell toxicity assay
The samples' cytotoxicity against Hela cells were evaluated by MTT assay. Hela cells were cultured in a 96-well plate with 5×103 cells per well, and grew about 15 h. Then, the cells were incubated in 100 μL of complete DMEM containing samples with different concentrations at 37 ℃ in a 5% CO2 atmosphere for 48 h. Fresh pure complete DMEM was used as the control. After 20 μL of 5 mg/mL MTT solution was added in, the cells were continued to be incubated for 4 h. Finally, the medium in each well was then replaced with 150 μL of dimethyl sulfoxide. The plates were shaken for 10 min to ensure formazan crystals to be dissolved completely. The absorbance at a wavelength of 490 nm was recorded in a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The cell viability is calculated based on Eq.(3):
Here, Asample and Acontrol represent the absorbance of the sample solution and the control solution, respectively.F. Nuclease protection assay
The stability of pEGFP/CS complex particles was evaluated by agarose gel electrophoresis. pEGFP/CS complex particles containing 2 μg of pEGFP were firstly incubated in DNase Ⅰ (130 U/mL, 0.65 U/μg DNA) at 37 ℃ for 10 min. 2 μL of EDTA solution (0.5 mol/L) was added to stop the digestion effect of DNase Ⅰ. EB (10 μg/mL) was dissolved in the hot agarose solution (1%, w/v). The system was cooled to room temperature to form an agarose gel. The agarose gel was put into a TAE buffer. The samples were injected in the gel for the electrophoresis analysis at 100 V for 30 min. Finally, the gel was observed under UV light.G. In vitro gene transfection of pEGFP/CS-X complex particles
Hela cells were seeded onto 96-well culture plates with 5×103 cells per well. Cells were incubated in complete DMEM medium (200 μL/well) at 37 ℃ for 20 h in a humidified 5% CO2 incubator (Dan Ding Shanghai International Trade Co., Ltd., Shanghai, China). The medium was then replaced by 100 μL/well of serum-free medium containing pEGFP/CS-X or pEGFP/PEI complex particles. After that, cells were continued to be incubated for 4 h at 37 ℃. The mediums were all replaced with complete DMEM (200 μL/well) for further transfection. 100 μL of fresh serum-free DMEM was used as the blank control. After 72 h's incubation, the complete medium in each well was replaced by PBS solution (10%, 100 μL/well). Then pEGFP expression was qualitatively evaluated by fluorescence microscope (Olympus, Tokyo, Japan), and quantitatively measured by flow cytometer (BD FACSVerse, BD, Franklin Lakes, NJ, USA).Ⅲ. RESULTS AND DISCUSSION A. The γ-ray radiation effect on CS in acetate acid solution
Figure 1 shows the change in the appearance of the acetate acid solution of CS after γ-ray radiation at different absorbed dose. It is clearly seen that the colors of the CS solutions change from colorless to yellow after γ-ray radiation, and darken with the increase of the absorbed dose. The result indicates that there are some changes on the chain structure of CS after γ-ray radiation. It is well known that CS is a copolymer consisting of glucosamine and N-acetylglucosamine units linked by β (1-4) glycosidic bonds. When CS was subjected to γ-ray radiation, chain scissions may occur through the breakage of β (1-4) glycosidic bonds. The change in the colour of CS solution during the irradiation process has also been reported in previous literatures [46, 47], which was attributed to the carbonyl and carboxyl groups produced by some oxidations happening during the irradiation process.
The FT-IR spectra of CS-X are displayed in Fig. 2, which are similar to the spectrum of CS-0. But the enhancement on the characteristic absorbance for the stretching vibration of C=O at 1720 cm-1 indicates the generation of carbonyl and/or carboxyl groups after γ-ray radiation.
The element analysis for all CS-X samples listed in Table Ⅰ can also confirm the breakage of C-N bonds. The molar ratio of N/C of CS has a slight fall after CS was irradiated by γ-ray, and has little dependence on the absorbed dose lower than 50 kGy. This means the content of N element in CS-X could be regarded as a constant, and has little influence on N/P ratio in the formation of pEGFP/CS-X complex particles.
The change of the MW of CS radiated at different absorbed doses measured by Ubbelohde viscometry is exhibited in Fig. 3. It is clearly seen that the MW of CS decreases dramatically with the increase of the absorbed dose, indicating the degradation of CS chains under the γ-ray radiation. When the absorbed dose is above 30 kGy, the MW will decrease about an order of magnitude, i.e. from the original 3.5×10-5 g/mol to 9.0×10-4 g/mol (30 kGy), and 5.0×10-4 g/mol (50 kGy). The changes in MW of the CS-irradiated indicate that CS is a kind of polymer easy to be scissioned by high energy radiation [47, 54].B. Cell toxicity assays of the γ-ray-radiation-scissioned CS
Biosafety is a basic requirement for gene transfection vectors. The MTT assays of the γ-ray-radiation-scissioned CS have been investigated, and the results are shown in Fig. 4. As a comparison, the cell toxicity of a commonly studied synthetic cationic vector, PEI, is also displayed in Fig. 4. It is seen that the natural CS (CS-0) shows excellent biosafety since the cell viability in the presence of CS is nearly as high as 100% at a concentration up to 10 μg/mL. At a relatively low concentration (0.5 μg/mL), all of the γ-ray-radiation-scissioned CS samples also exhibit low cell toxicity. The cell viabilities are 99.9% of CS-10, 94.3% of CS-30, and 92.1% of CS-50, respectively, much higher than that of PEI (61.6%). The cell toxicity of the γ-ray-radiation-scissioned CS samples increases slightly with their concentration, but the cell viability in the presence of 10 μg/mL of each γ-ray-radiation-scissioned CS samples remains above 87.5%, which demonstrates that the γ-ray-radiation-scissioned CS has little hindrance on the growth of Hela cells. The slight increase of the cytotoxicity is probably caused by the carbonyl and carboxyl groups generated under γ-ray radiation of CS . However, under the same conditions, the cell toxicity of PEI rises rapidly with its concentration. When the concentration increases to 1 μg/mL, more than a half of the cells die (the cell viability is only 48.2%). When the concentration of PEI increases to 10 μg/mL, only 11.9% of the cells survives. These results show the low-MW CS prepared by γ-ray-radiation-scission method still has much higher biosafety than the current promising synthetic PEI gene carrier.C. Characterization of pEGFP/CS-X complex particles
The plasmid (pEGFP) and γ-ray-radiation-scissioned CS can form complex particles through the complex coacervation method since the unit structure of CS has little change under γ-ray-radiation. The particle size and size distribution of pEGFP/CS-X complex particles were measured by DLS, as shown in Fig. 5 and Table Ⅱ. It reveals that the size of the pEGFP/CS-X complex particles is about 200-300 nm, which is favorable for the in vivo transportation and endocytosis . At the same time, the size of pEGFP/CS-X complex particles reduces slightly with the MW of CS.
Zeta potentials of pEGFP/CS-X complex particles can be also measured by DLS and listed in Table Ⅱ. The Zeta potentials of pEGFP/CS-X complex particles are all positive, and a little slight decrease with the MW of CS. This may be related with the lower entanglement degree of low-MW CS chains. Since the complex of pEGFP and CS is driven by the electrostatic attraction, the lower chain entanglement makes less structure units on CS chains be attracted around pEGFP at the same N/P ratio so that the complex particles have a lower positive Zeta potential, as well as a smaller particle size.
The protection effect of CS-X on the loaded pEGFP was investigated since the stability of the gene vectors during the in vivo circulation is one of the key issues for gene transfection. Figure 6 exhibits gel electrophoresis of pEGFP/CS-X complex particles before and after being digested by DNase Ⅰ. Figure 6(a) proves that the pEGFP can be combined stably with either natural CS or γ-ray-radiation-scissioned CS since there is no outside bright bands for all pEGFP/CS-X complex particles but the naked pEGFP. After being treated with DNase Ⅰ, all the naked pEGFP disappears by digestion and no light band of naked pEGFP can be observed (Fig. 6(b)). At the same time, no changes happened for all of CS-X groups, indicating that all of the CS-X possess an effective protection effect on the loaded pEGFP.D. In vitro gene transfection of pEGFP/CS-X complex particles
The transfection efficiencies of pEGFP/CS-X complex particles were investigated in DMEM with PEI as a positive control and a blank group as a negative control. The results are observed by fluorescence microscope, as shown in Fig. 7.
Under the fluorescence microscope, the blank group shows almost no fluorescence, i.e. there is no transfection of pEGFP as so to no expression of EGFP. While green fluorescence can be clearly observed for all pEGFP/CS-X groups, and the intensity of green fluorescence seems much stronger than that for pEGFP/CS-0 group, indicating that using the low-MW CS obtained by γ-ray-radiation-scission method as the gene carrier benefits to achieve higher transfection efficiency. Furthermore, the transfection efficiency will be improved when CS was irradiated at a larger absorbed dose.
The PEI group shows the highest fluorescence intensity. However, from the white field, it is clearly seen that Hela cells in PEI group are totally different from the other groups. As proven in Fig. 4, PEI has much higher cytotoxicity than all of CS-X samples. It will kill a part of cells, and at the same time, make those survivals unhealthy (contraction and distortion). On the other hand, the cells in other groups seem to be plump and healthy.
The quantitative assay for in vitro gene transfection efficiency was evaluated by flow cytometry, as shown in Fig. 8. Fluorescence activated cell sorting (FACS) results are in accord with the results of Fig. 7, which demonstrates that the gene vectors using γ-ray-radiation-scissioned CS as the carrier will possess better gene transfection efficiency than those using natural high-MW CS as the carrier. The higher the absorbed dose, the smaller the MW of CS and the better transfection efficiency of the corresponding gene vector.Ⅳ. CONCLUSION
In summary, low-MW CS can be prepared through the γ-ray radiation on the acetic solution of CS. The CS chains were scissioned under the γ-ray radiation, and the MW of CS decreased with the absorbed dose. When the absorbed dose was above 30 kGy, the MW decreased about an order of magnitude, i.e. from the original 3.5×10-5 g/mol to 9.0×10-4 g/mol (30 kGy) and 5.0×10-4 g/mol (50 kGy). The cell toxicity of the γ-ray-radiation-scissioned CS increased slightly compared with the original CS, but can still reach a cell viability as high as above 87.5%. The γ-ray-radiation-scissioned CS can be combined with plasmid (pEGFP) through complex coacervation method, forming pEGFP/CS-X complex particles with a size of 200-300 nm. The pEGFP/CS-X complex particles have a good stability and little cytotoxicity. The in vitro gene transfection efficiency of the pEGFP/CS-X complex particles were investigated by fluorescence microscope and flow cytometry. The results showed that the gene vectors using γ-ray-radiation-scissioned CS as the carrier will possess better gene transfection efficiency than those using natural high-MW CS as the carrier. The higher the absorbed dose, the smaller the MW of CS, and the better transfection efficiency of the corresponding gene vector.Ⅴ. ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China (No.81171829, No.51473152, and No.51573175) and the Fundamental Research Funds for the Central Universities (WK2060200012, WK3450000001). We also thank Prof. Li-hua Yang and Prof. Ye-zi You at the University of Science and Technology of China (USTC) for their kind help in providing experimental reagents and instruments.
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b. 安徽医科大学第一附属医院整形外科, 合肥 230031