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TAT-functionalized PEI-grafting rice bran polysaccharides for safe and efficient gene delivery
International Journal of Biological Macromolecules xxx (xxxx) xxx
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
TAT-functionalized PEI-grafting rice bran polysaccharides for safe and efficient gene delivery Liang Liu a,1,⇑, Yujian Yan a,1, Danni Ni a, Shuheng Wu a, Yiran Chen a, Xin Chen a, Xuemin Xiong a, Gang Liu b,⇑ a b
School of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, China College of Food and Science Engineering, Wuhan Polytechnic University, Wuhan 430023, China
a r t i c l e
i n f o
Article history: Received 12 August 2019 Received in revised form 5 September 2019 Accepted 22 September 2019 Available online xxxx
a b s t r a c t Polysaccharides are considered to be promising candidates for non-viral gene delivery because of their molecular diversity, which can be modified to fine-tune their physicochemical properties. In this work, transcriptional activator protein (TAT) functionalized PEI grafted polysaccharide polymer (PRBP) was prepared by using rice bran polysaccharide as the starting material, and characterized by various methods. The potential of TAT functionalized PRBP (PRBP-TAT) as gene vector was studied in vitro, including DNA loading capacity, DNA protection ability and biocompatibility. The cell uptake and transfection efficiency of the PRBP-TAT/pDNA polyplexes were studied. The results showed that PRBP-TAT could completely condense DNA at N/P 2. The PRBP-TAT/pDNA polyplexes could protect DNA from degrading by DNase and were efficiently internalized by cells. Biocompatibility result showed that PRBP-TAT had no significant cytotoxicity and effect on cell proliferation. At low N/P ratios of 1–3.5, PRBP-TAT showed higher transfection efficiency than PEI30k and PEI30k-grafted rice bran polysaccharide. PRBP-TAT and PEI showed the highest transfection efficiency of 42.8% and 28.1% when pDNA is 2 mg and N/P ratio is 1.5, respectively, while PRBP showed the highest transfection efficiency of 37.3% at N/P 2.5. These results indicate that PTA is a promising candidate vector for safe and efficient gene delivery. Ó 2019 Elsevier B.V. All rights reserved.
1. Introduction As a new cancer treatment strategy, gene therapy has attracted much attention because of its remarkable therapeutic efficacy [1]. However, safe and efficient gene delivery is still one of the main obstacles in gene therapy. Viral gene vectors have been extensively studied due to their efficient transfection efficiency [2]. However, since viral vectors have serious defects including inflammation response, production difficulties, inflammatory reactions, immunogenicity, and carcinogenicity, they are rarely used in actual clinical treatment [3]. Although the gene transfection efficiency of the non-viral vector is lower than that of the viral vector, the toxicity and immunogenicity are lower than the viral vector [4]. Thus, such as liposomes, micelles, cationic polymers, and polyanionic polymers, etc., have been extensively studied to replace viral vectors for efficient gene delivery [5]. Among various non-viral gene carriers, cationic polymers have attracted the attention of researchers due to their unique physicochemical properties [6]. PEI is one of the most widely studied materials as gene carriers [7]. PEI performs high DNA condensation ⇑ Corresponding authors. 1
E-mail addresses: [email protected] (L. Liu), [email protected] (G. Liu). These authors contributed equally to this work.
ability and strong proton sponge effect, which can destroy the endolysosome and internalize the vector/pDNA complex into the cytoplasm, and is therefore considered to be the gold standard for evaluating transfection efficiency [8]. However, as a gene delivery vector, PEI has several problems to be solved: such as severe cytotoxicity of PEI caused by excessive cationic charge [9,10]. Therefore, researchers modified PEI by various biodegradable materials to address this issue [11,12]. Polysaccharides have a variable molecular structure due to the variability of monomers and the stereospecificity of glycosidic linkages and differences in monosaccharide sequences [13]. Polysaccharides have many advantages as biomaterials, such as excellent biocompatibility and biodegradability, which can be obtained from agricultural or marine supplementary food resources [14,15]. Therefore, polysaccharide is widely used to modify PEI to prepare a low toxicity, biodegradable gene and drug vector [16]. Rice bran polysaccharides (RBP) has a wide range of biological activities, including anti-tumor, antioxidant and anti-inflammatory effects, as well as improved diarrhea and immune function [17,18]. So far, RBP is rarely studied as a biological material, especially as drug and gene delivery vector. In previous study, we have synthesized low molecular weight PEI (Mw = 600, 2000 Da) modified RBP (PEI-RBP), and the PEI-RBP exhibited higher transfection efficiency and better biocompatibility than PEI600 and PEI2k. But
https://doi.org/10.1016/j.ijbiomac.2019.09.234 0141-8130/Ó 2019 Elsevier B.V. All rights reserved.
Please cite this article as: L. Liu, Y. Yan, D. Ni et al., TAT-functionalized PEI-grafting rice bran polysaccharides for safe and efficient gene delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.234
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in comparison with commercial transfection reagents of lipo6000, the gene transfection efficiency of PEI-RBP is still unsatisfactory [19]. TAT (transcriptional activator protein) is a short, positively charged peptide that helps macromolecules enter mammalian cells [20]. TAT peptide, containing 11-amino-acid within the HIV-1 TAT protein, is able to act as nuclear localization signal domain and easily translocate both the plasma membrane and the nuclear membrane into the cellular nuclear [21]. Recent studies have shown that many substances conjugated with TAT can penetrate cell membranes and perform their biological functions [22]. TAT has also been used to enhance the efficiency of gene transfection mediated by polysaccharides gene vector [23]. Tang et al. [24] proved that TAT peptide-conjugated hyaluronic acid could condense DNA, and the vector/pDNA polyplexes could be able to efficiently release DNA and deliver DNA into the nuclear, resulted in higher transfection efficiency than PEI/pDNA polyplexes. Yan et al. [25] reported the TAT peptide-conjugated N-succinylchitosan carriers for safe and efficient gene transfection and in vitro. On this basis, we hypothesize that the gene transfer efficiency of PEI-RBP can be further improved by grafting TAT, and a new efficient gene transfer system is expected to be discovered. In this study, in order to develop safe and efficient gene delivery vector based on RBP, PEI30k modified rice bran polysaccharides (PRBP) and TAT conjugated PRBP (PRBP-TAT) were prepared. The structure and chemical composition of PRBP-TAT were analyzed by FTIR, 1H NMR and elemental analysis. DNA binding and protection ability, biocompatibility of PRBP-TAT and DNA release, cell uptake and transfection efficiency of PRBP-TAT/pDNA were studied in vitro. This work is the first effort to improve the efficiency of PRBP-mediated gene transfection through TAT conjugation, and provide insights for the research and development of RBP as a biomaterial. 2. Materials and methods 2.1. Materials Rice bran (RB) was obtained from Hubei Hunsen Biotechnology Co., Ltd (Hubei, China) and the rice bran polysaccharides (RBP) were prepared and determined according to our previous work
[26]. Commercial branched polyethylenimines with average Mw 30 k Da (bPEI30k) was purchased from Macklin Inc (Shanghai, China). TAT (YGRKKRRQRRRC[NH2]) was purchased from Ontores Biotechnology Co., Ltd (Hangzhou, China). Lipo6000 transfection agent was gained from Beyotime Biotechnology (Shanghai, China). PikoGreen dsDNA quantitative Kit was purchased from Shanghai Liji Biotechnology Co., Ltd (Shanghai, China). The EGFP plasmid was amplified in E. coli, then extracted and purified by a plasmid extraction kit (Omega Bio-Tek, Guangzhou, China) according to the manufacturer’s protocol. 3-(4,5-dimethylthiazol-2-yl)-2,5-dip henyltetrazolium bromide (MTT), 4,6-diamidino 2-phenylindole (FITC) wase purchased from Sigma-Aldrich (Shanghai, China). Fetal bovine serum (FBS) and other cell culture medium were purchased from Gibco (New York, USA). Human embryonic kidney cells (293T), rat hepatocytes (BRL) and liver hepatocellular cells (HepG2) were purchased from National infrastructure of cell line resource (Beijing, China). Other biochemical reagents are analytical pure and purchased from Sinopharm Co., Ltd (Shanghai, China). 2.2. Preparation of PRBP and PRBP-TAT PRBP and PRBP-TAT were prepared according to the scheme in Fig. 1. PRBP: 500 mg RBP was reacted with 500 mg CDI in 150 mL DMSO containing 2 mL triethylamine for 3 h at rt, under nitrogen atmosphere, and protected from light. 50 mg PEI30k was then dissolved in 20 mL DMSO and dropped to the reaction mixture. After 24 h, the mixture was centrifuged, and the supernatant was collected and dialyzed in a dialysis bag to remove small molecular impurities. Three times of absolute ethanol was added to the dialysis interception solution, and the mixture was stayed overnight at 4 °C. Then precipitation was collected by centrifugation and pure PRBP was obtained by freeze-drying. The successful preparation of PRBP was confirmed by FTIR (Nicolet Avatar-370, Thermal Fisher Scientific, USA) and 1H NMR (Ascend400, BRUKER, Switzerland). PRBP-TAT: 100 mg PRBP and 100 mg CDI were reacted in 50 mL DMSO containing 2 mL triethylamine in a nitrogen atmosphere for 3 h. 10 mg TAT was dissolved in 5 mL DMSO, and then added to the above reaction solution, and the reaction was continued for 24 h. Dialysis (molecular weight cutoff, 3000 Da) was carried out to remove redundant TAT, and the dialyzate was freeze-dried to
Fig. 1. The preparation scheme of PRBP and PRBP-TAT.
Please cite this article as: L. Liu, Y. Yan, D. Ni et al., TAT-functionalized PEI-grafting rice bran polysaccharides for safe and efficient gene delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.234
L. Liu et al. / International Journal of Biological Macromolecules xxx (xxxx) xxx
obtain PRBP-TAT. The successful preparation of PRBP-TAT was confirmed by FTIR and 1H NMR.
2.3. Preparation of FITC labeling PRBP and PRBP-TAT Fluorescein isothiocyanate (FITC) labeling PRBP (FITC-PRBP) and PRBP-TAT (FITC-PRBP-TAT) were synthesized to evaluate the cellular uptake efficiency of vector/pDNA polyplexes. 5 mg PRBP and 0.2 mg FITC were reacted in deionized water at rt for 24 h. Then the dialysis assay was used to remove the free FITC, and the FITC-PRBP was obtained by freeze-drying. FITC-PRBP-TAT was prepared in the same method. FITC-PRBP and FITC-PRBP-TAT were characterized by fluorescence spectroscopy (LS55, PerkinElmer, USA) and UV–Vis spectroscopy (Lambda 25, PerkinElmer, USA).
2.4. Characterization The prepared PRBP and PRBP-TAT were characterized by FTIR (Fourier transform infrared spectroscopy), 1H NMR and elemental analysis. For FTIR analysis, 3 mg PRBP or PRBP-TAT was mixed with 600 mg potassium bromide (KBr) in a mortar, and then used to press tablet. A infrared spectrometer (PerkinElmer Frontier, PerkinElmer, USA) was used to measured the absorption at wave number range of 600–4000 cm 1. For 1H NMR, 10 mg PRBP and PRBPTAT was dissolved in 1 mL D2O, respectively, then loaded into a nuclear magnetic resonance tube for analysis (Ascend 400, Bruker, USA). Elemental analysis was carried out on a FLASH 2000 NC analyzer (Thermo Fisher Scientific, USA).
2.5. Preparation of vector/pDNA polyplexes The PRBP-TAT/pDNA polyplexes with various N/P ratios were prepared for different assays. EGFP plasmid DNA and different dosage PRBP-TAT were incubated in DMEM solution at 37 °C for 30 min to obtain the PRBP-TAT/pDNA polyplexes. The polyplexes contain 0.2 lg EGFP pDNA was used to prepare the PRBP-TAT/ pDNA polyplexes for gel retardation assay, and 1 lg for cell uptake and gene transfection assays. The vector/pDNA polyplexes DMEM solution was mixed by vortex before use. The PRBP/pDNA polyplexes were prepared in the same way, but PRBP is used instead of PRBP-TAT. The vector/pDNA polyplexes were dispersed in 0.001 M KCl solution and their particle size and zeta potential were determined by a Zetasizer Nano S90 (Malvern, UK).
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2.7. Cell culture 293T, BRL and HepG2 cells were used in the MTT, RTCA, cell uptake and EGFP gene transfection assays. Cells were cultured in high glucose DMEM supplemented with 10% (v/v) heatinactivated fetal bovine serum (FBS) and 1% antibiotic agent (penicillin and streptomycin) at 37 °C in a 5% CO2 atmosphere. 2.8. MTT and RTCA assay The cytotoxicity of PRBP-TAT was evaluated by MTT assay in 293T, BRL and HepG2 cells. Cells were seeded in 96-well plate at density of 1 104 cells/well and cultured in complete medium for 24 h. Then the culture medium was removed and various concentrations (0.005, 0.01, 0.015, 0.02, 0.025 and 0.03 mg/mL) PRBPTAT were added to cells, respectively. After incubation for 36 h, the medium was removed and the cells were washed by PBS for three times, and then the cells were treated with 10 mL 0.5% (m/v) MTT DMEM solution for 4 h. 150 mL DMSO was then used to replace the MTT solution and dissolve the formazan crystals. The absorbance of each well at 490 nm (A490) was measured by a microplate reader (EnspireTM, PerkinElmer, USA). The cells treated with DMEM were set as blank and six replicates were made for each concentration of the PRBP-TAT. The cytotoxicity of PRBP-TAT/pDNA polyplexes were studied as the same method. PRBP-TAT/pDNA polyplexes were prepared according to Section 2.5, and the N/P ratios were 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5. For comparison, the cytotoxicity of PEI30k, PEI30k/pDNA, PRBP and PRBP/pDNA polyplexes was also studied. RTCA is based on the analysis of cell activity related to the growth trend of adherent cells via an xCELLigence RTCA S16 device (ACEA Biosciences, Inc., Hangzhou, China). RTCA detects the effect of cell adherence on the electric resistance of an e-plate which is consistent with cell viability in real time. RTCA assay was performed according to the manufacturer’s instructions. 200 mL complete medium was added into each well of the e-plate to calibrate the instrument baseline. Then, cells were seeded into each well and cultured in 5% CO2 at 37 °C for 30 min. The e-plate was placed on the detector and culturing continued for 24 h until the cell concentration was approximately 70–80%. After that, the culture medium was removed, complete media containing different concentrations of PRBP-TAT were added into each well, respectively, and the cells were continually cultured in 5% CO2 at 37 °C for 48 h. The cell index data were recorded every 5 min during the beginning 2 h, then per 2 h during the later stage. The data were exported after the end of experiment through a manufacturer’s software of RTCA iCELLigence V2.0.
2.6. DNA release 2.9. Cell uptake assay Vector/DNA polyplexes were prepared according to Section 2.5. The reaction mixture was centrifuged to remove the DMEM and free DNA. Then the vector/DNA polyplexes were dispersed in 5 mL PBS (phosphate buffer solution, pH 7.4) and sodium acetate buffer solution (pH 5.0), respectively, and the solution was shaken at 100 rpm at 37 °C in a biochemical incubator. To study the DNA release, the suspension was centrifuged after various time, then 10 lL supernatant was removed and the amount of DNA was measured by a PikoGreen dsDNA quantitative Kit according to the manufacturer’s instructions. After each measurement, the total volume of suspension was supplemented with DMEM to 5 mL to keep the total volume unchanged. The DNA release ratio was calculated as: DNA release ratio = (m1/m0) 100%. m1 is the amount of DNA in vector/DNA polyplexes, m0 is the amount of releasing DNA from vector/DNA polyplexes. Vector represents PRBP or PRBP-TAT in this section. The standard error of the mean was calculated from the three independent measurements.
Cell uptake is one of the main obstacles in the process of gene delivery by non-viral vectors. Effective cell uptake is the prerequisite for efficient gene transfection. In this work, the uptake efficiency of PRBP-TAT/pDNA and PRBP/pDNA polyplexes by 293T cells were investigated by fluorescence microscope and flow cytometry. In order to realize cell uptake results visualization, FITC labeled PRBP-TAT and PRBP were used to form the vector/pDNA polyplexes. The N/P ratios were 1 to 3.5 for FITC-PRBP/pDNA and FITC-PRBP-TAT/pDNA polyplexes. 293T cells were seeded in 12well plate at a density of 1 105 cells/well and cultured in complete medium until the cell density reached 70–80%. Then the DMEM dispersion of FITC-vector/pDNA polyplexes with various N/P ratios was used to replace the the culture medium, respectively, and the cells were cultured for 4 h. Cells were then fixed with 1% paraformaldehyde and observed by an inverted fluorescence microscope (ix53, Olympus, Japan). Moreover, the cellular
Please cite this article as: L. Liu, Y. Yan, D. Ni et al., TAT-functionalized PEI-grafting rice bran polysaccharides for safe and efficient gene delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.234
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uptake efficiency of FITC-vector/pDNA polyplexes was quantitatively measured by flow cytometer (FACSCanto II, BD, USA). The data are given as mean ± standard deviation (SD) based on 3 measurements. 2.10. In vitro gene transfection study In order to investigate the influence of pDNA doses on transfection efficiency, vector/pDNA polyplexes with the same N/P ratio were formed with EGFP plasmid DNA of 1 and 2 ug, respectively, according to Section 2.5. The N/P ratios were 1 to 3.5 for PRBP/ pDNA, PRBP-TAT/pDNA, and PEI30k/pDNA. Lipo6000 was used as positive control and naked DNA as blank control. For lipo6000 mediated plasmid transfection, 1.0 mg plasmid DNA and 2.0 mL lipo6000 transfection reagent were added in 5 mL DMEM, respectively, then mixed and incubated at room temperature for 10 min before transfection. The ratio of plasmid (mg) to Lipo6000 (mL) was 1:2 (m/v). 293T cells were seeded in 24-well plates with density of 3 l04 cells/well and cultured in complete medium until
reached 70–80% confluence, and then the medium was removed and cells were washed with PBS. The vector/pDNA polyplexes were slowly added to the cells and cultured for 4 h. Then 1 mL complete medium was used to replace the primary medium and cultured for 48 h. The transfection result was observed by fluorescence microscope and quantitatively measured by flow cytometer. For flow cytometer assay, the transfected cells were digested by trypsin and washed by PBS, then dispersed in the working fluid and the positive expression EGFP cells were measured by flow cytometer. The data are given as mean ± standard deviation (SD) based on 3 measurements. 3. Results 3.1. Characterization The 1H NMR spectrum was used to confirm the successful preparation of PRBP and PRBP-TAT. The 1H NMR spectra of RBP (A), PEI30k (B), PRBP (C) and PRBP-TAT (D) are shown in Fig. 2.
(A)
(B)
RBP
PEI30k
(D)
(C) PRBP
PRBP-TAT
(E)
Fig. 2. The 1H NMR spectra of RBP (A), PEI30k (B), PRBP30k (C), PRBP-TAT (D) and the FTIR spectra of RBP, PRBP and PRBP-TAT.
Please cite this article as: L. Liu, Y. Yan, D. Ni et al., TAT-functionalized PEI-grafting rice bran polysaccharides for safe and efficient gene delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.234
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H NMR usually provides the information of glycoside bond configuration and hydrogen number ratio in polysaccharide structure. However, due to the similar chemical shifts of non-heteropoly H and partial overlap, most H signals are difficult to analyze. The chemical shifts of general b heteropoly region are in the range of d 4.4 – 5.0, and those of a heteropoly region are in the range of d 5.0 – 5.5. The 1H NMR signals of RBP have d 5.209 and 4.613 which indicate that there are two glycoside configurations of a and b in RBP. The peaks at 2.4–2.6 ppm in the NMR spectra of PEI30k, PRBP and PRBP-TAT copolymers correspond to –NHCH2CH2– in PEI, which indicates that PEI is conjugated to RBP. The peak at 1– 2 ppm is attributed to –CH2– of TAT, which appears in the PRBPTAT spectrum, indicating that the TAT peptide was successfully coupled to PRBP. The FTIR spectra of RBP, PRBP and PRBP-TAT are shown in Fig. 2E. For RBP, the peak of 3200–3400 cm 1 is caused by the stretching vibration of –OH, the peaks near 2390 cm 1 are caused by C–H vibration. Compared with RBP, the PRBP FTIR spectra shows that the peak intensity at 3200–3400 cm 1 is decreased, which is due to the reducing of hydroxyl caused by the PEI conjugation. The peak of 1600 cm 1 contributes to vibration of N–H, the peak of 1000–1200 cm 1 contributes to the characteristic peak of C–O–C, the peak of 1300 cm 1 is caused by the stretching vibration of C–N, and peak of 1570 cm 1 is caused by vibration of N–H. These results indicate that the grafting of PEI30k onto RBP is successful. The peak at 1450–1650 cm 1 is the absorption peak of tyrosine benzene ring in TAT peptide. This indicates that the successfully TAT grafting of PRBP. The contents of C, N and H of PRBP and PRBP0-TAT were studied by elemental analysis. As shown in Table 1, the result exhibits that the content of N and C slightly decreases from PRBP to PRBP-TAT, while H increases by 2%. This is because the percentage of N and C in PRBP is higher than that in TAT, while the content of H is lower than that in TAT. The N/P ratios of PRBP-TAT/pDNA and PRBP/ pDNA polyplexes are calculated according to the elemental analysis results. The elemental analysis further proves that TAT is successfully grafted to PRBP.
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3.2. Particle size and zeta potential of vector/pDNA polyplexes To determine the particle size of vector/pDNA polyplexes, pDNA was complexed with PRBP or PRBP-TAT at various N/P ratios of 1– 3.5, then analyzed using DLS. As shown in Fig. 3A, the particle size of vector/pDNA polyplexes were decreasing with the increase of N/P ratio. At N/P 3.5, the PRBP/ pDNA and PRBP-TAT/pDNA polyplexes have a average particle size of less than 200 nm and are suitable for cellular uptake. PRBP-TAT/pDNA polyplexes have a smaller particle size than PRBP/pDNA at different N/P ratios. This is due to the fact that the charge density of PRBP-TAT (Fig. 3B) is higher than PRBP and thus has a stronger DNA condensation ability. This means that the zeta potential of PRBP-TAT benefits from the TAT conjunction. Moreover, the result (Fig. 3B) shows that the zeta potential of various vector/pDNA polyplexes is increased relative to N/P ratios. 3.3. DNA loading and protection Naked DNA can be degraded by DNase I, and enzymatic degradation contributes to a major limitation for gene delivery. Therefore, DNA condensation and protection ability is one of the basic capabilities for non-viral gene vectors. The interactions of RBP based vectors with ctDNA were investigated by agarose gel electrophoresis. As shown in Fig. 4A, the results show that PEI30k, PRBP and PRBP-TAT can condensate DNA at low N/P ratio. The minimum N/P ratio which is needed to condensed DNA is 1.5 for PEI30k, 2.5 for PRBP and 1 for PRBP-TAT. This is because the conjugation of RBP decreases the positive charge density of PEI30k, while the modification of TAT increases the positive charge density of PRBP. The protective ability of PEI30k, PRBP and PRBP-TAT to DNA is shown in Fig. 4B. The results showed that after incubation with DNase I for 4 h, the naked pDNA was completely degraded, but the DNA present in the form of vector/DNA polyplex (which was blocked in the loading well) was not degraded. These results indicate that PRBP-TAT can effectively condense and protect DNA from enzymatic degradation. 3.4. DNA release
Table 1 The contents of C, N, H in PRBP and PRBP-TAT. Element
Content (%) in PRBP
Content (%) in PRBP-TAT
C N H
46.78 24.85 8.10
44.36 23.59 10.10
The DNA release was evaluated by PicoGreen kit. As shown in Fig. 5, the release profile of plasmid from vector/pDNA polyplex displays a biphasic pattern that was characterized by a first rapid release within 2 h, followed by a slower and sustained release within 10 h, at pH 7.4. The release of the plasmid rate of PRBP/ pDNA and was somewhat slower than those of PRBP-TAT/pDNA. The results exhibit that PRBP-TAT/pDNA not only has a higher
Fig. 3. Particle size (A) and zeta potential (B) of vector/pDNA polyplexes.
Please cite this article as: L. Liu, Y. Yan, D. Ni et al., TAT-functionalized PEI-grafting rice bran polysaccharides for safe and efficient gene delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.234
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(A)
(B)
positively correlated with their charge density. RBP modification reduces the charge density of PEI, while TAT grafting increases the charge density of PRBP. The cytotoxicity of vector/pDNA polyplexes with various N/P ratio was shown as Fig. 6D. It shows that PRBP-TAT/pDAN polyplexes show lower cytotoxicity than PEI30k/ pDNA and PRBP/pDNA. These results suggest that TAT functionalization increases the cytotoxicity of PRBP, but doesn’t effect the PRBP-TAT/pDAN polyplexes exhibit a good biocompatibility. This is due to DNA neutralizing the positive charge density of PRBPTAT in polyplexes (Fig. 3B). RTCA assay was used to analyze the effects of vectors on cell growth on-line. As Fig. 6E and F show, PRBP and PRBP-TAT manifest a slight inhibitory effect on the growth of BRL and HepG2 cells, while PEI30k shows more significant inhibitory effect on cell growth. This is consistent with the results of MTT assay, and it is asserted that PRBP-TAT has a better biocompatibility than PEI30k. 3.6. Cell uptake
Fig. 4. DNA binding (A) and protection (B) ability of PEI30k, PRBP and PRPB-TAT.
Fig. 5. DNA release ratio of PRBP/pDNA and PRBP-TAT/pDNA polyplexes at various pH condition. Results are represented as mean ± SD (n = 3).
DNA release rate, but also a high release ratio at pH 5.0 than PRBP/ pDNA. This suggests that PRBP-TAT/pDNA pilyplexes can release DNA efficiently and may perform more efficient gene transfection than PRBP/pDNA.
The vector/pDNA polyplexes with fluorescent probes were prepared by FITC-labeled vectors. Then the cell absorption of the polyplexes was studied by fluorescence microscopy and flow cytometry, respectively. As shown in Fig. 7A and B, the UV–visible absorption spectrum and fluorescence emission spectrum of FITC labeled PRBP and PRBP-TAT had similar characteristic peaks with FITC, but they were completely different before labeling. The slight red shift in the labeled PBP and PBP-TAT spectra is attributed to the change of pH caused by amino group in PEI. This results indicate that PRBP and PRBP-TAT are successfully labeled by FITC. The DNA condensation ability of FITC labeled PRBP and PRBP-TAT was examined by gel retardation assay. The result shows (Fig. 7C) that FITC-PRBP and FITC-PRBP-TAT can completely condensate DNA at N/P 2 and 1, respectively. Therefore, it is feasible to use FITC to label the vector to study the cellular uptake of the vector/pDNA polyplexes. An inverted fluorescence microscopy was used to observe the cell uptake of the labeled vector/pDNA polyplexes. As shown in Fig. 7D, after 4 h of treatment with various N/P ratios of FITClabeled vectors/pDNA polyplexes, obvious green fluorescence was observed in the cells, and the fluorescence intensity increases with the increase of N/P. This is attributed by the decrease of particle size of polyplexes caused by the increase of N/P (Fig. 3A) [5]. The results manifest that the polyplexes can be efficiently absorbed by cells. Flow cytometry (Fig. 7E) results confirmed that PRBPTAT/pDNA and PRBP/pDNA polyplexes could be internalized in cells, and the uptake efficiency increased with the increase of N/P ratio. It showed that the uptake efficiency of PRBP-TATt/pDNA was 95% at N/P 3.5, while that of PRBP/pDNA was 65%. In the N/P range of 1–3.5, PRBP-TAT/pDNA polyplexes exhibited higher the uptake efficiency than PRBP/pDNA, indicating that the cell uptake efficiency of PRBP was improved by the TAT peptide functionalization. 3.7. In vitro gene transfection study
3.5. Biocompatibility MTT assay was used to evaluate the cytotoxicity of PRBP-TAT and PRBP-TAT/pDNA to HepG2, BRL, and 293T cells. Cells were treated with various concentrations of PRBP-TAT, respectively. The concentration range of PRBP-TAT was consistent with that of uptake and gene transfection assays, ranging from 5 to 30 mg/mL. The cytotoxicity of PEI30k and PRBP was evaluated for comparison. Fig. 6A, B and C show that RBP is not cytotoxic to the three cells, conversely, PEI shows strong cytotoxicity. Different samples show the following sequence of cytotoxicity to cells: PEI > PRBP-TAT > P RBP > RBP. This is because the cytotoxicity of non-viral vectors is
PRBP-TAT-mediated gene transfection was carried out in 293T cells. 1 mg and 2 mg EGFP encoding plasmid DNA were used as the reporter gene, respectively. Cells treated with DMEM were set as blank control and commercial reagent lipo6000 was set as positive control. For comparison, the transfection mediated by PEI30k and PBRP were also evaluated. As Fig. 8A shows, after 24 h of treatment with different vector/pDNA polyplexes with N/ P ratios of 1–3.5, obvious GFP expression (green fluorescence) was observed in cells, but not in cells treated with naked DNA (data not show). Compared with PRBP/pDNA, PRBP-TAT/pDNA showed more significant GFP expression.
Please cite this article as: L. Liu, Y. Yan, D. Ni et al., TAT-functionalized PEI-grafting rice bran polysaccharides for safe and efficient gene delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.234
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Fig. 6. The biocompatibility results. (A) The cell viability of 293T, (B) BRL and (C) HepG2 cells after treated with various concentration of RPB, PEI30k, PRBP and PRBP-TAT. (D) The cell viability of 293T cells after treated with PEI30k/pDNA, PRBP/pDNA and PRBP-TAT/pDNA polyplexes with various of N/P ratios. The real time cell index of BRL (E) and HepG2 (F) cells after treated with various concentration of RPB, PEI30k, PRBP and PRBP-TAT. Results are represented as mean ± SD (n = 6).
Flow cytometry was used to quantitatively determine the transfection efficiency of different vectors. The transfection efficiency increases with the increasing of pDNA dosage, as shown in Fig. 8B, the transfection efficiency of vectors with 2 mg DNA was significantly higher than that with 1 mg. However, the transfection efficiency doesn’t further increase with the increasing of pDNA dosage (data not show). It shows that PRBP has higher transfection efficiency than PEI30k. When pDNA is 2 mg and N/P ratio is 2.5, the highest transfection efficiency of PRBP is 37.3%, while PEI shows the highest transfection efficiency of 28.1% at N/P ratio of 1.5. This may be due to the reduction of cytotoxicity of PEI30k by polysaccharide grafting, thus improving the transfection efficiency. Remarkably, PRBP-TAT exhibits higher transfection efficiency than both PEI30k and PRBP. It is shows that PRBP-TAT performs the highest transfection efficiency of 42.8% when pDNA is 2 mg and N/P ratio is 1.5. In addition, the disparity of transfection efficiency
between PRBP-TAT/pDNA and PRBP/pDNA was more significant when 1 mg pDNA was used for transfection experiments. Comparison of the cell uptake results, it can be inferred that the higher transfection efficiency of PRBP-TAT/pDNA is contributed to the higher cell uptake efficiency. This indicates that it is feasible to improve the transfection efficiency through TAT functionalization. 4. Discussion Polysaccharides are non-toxic, biodegradable and biocompatible materials [13]. They are particularly suitable for blood transfusion and biological applications, because they are water-soluble and can be easily transported to cells in vivo, thus serving as effective carriers for delivering drugs combined with them [27]. In addition, polysaccharides have molecular diversity and can be modified to regulate their physical and chemical properties, so they are con-
Please cite this article as: L. Liu, Y. Yan, D. Ni et al., TAT-functionalized PEI-grafting rice bran polysaccharides for safe and efficient gene delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.234
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Fig. 7. (A) The UV–Vis absorbance spectrum of FITC-PRPB and FITC-PRBP-TAT. (B) The fluorescence emission spectrum of FITC-PRPB and FITC-PRBP-TAT. (C) DNA condensation ability of FITC-PRPB and FITC-PRBP-TAT. (D) Fluorescence microscope images of 293T cells with positive FITC-PRBP/DNA polyplexes uptake, the cells images were observed by an inverted fluorescence microscope at a magnification of 40. Scale bar = 500 lm. (E) Flow cytometry results of cell uptake efficiency of FITC-PRPB/pDNA and FITC-PRBP-TAT/pDNA polyplexes. Results are represented as mean ± SD, (n = 3).
sidered as a promising non-viral gene vector [28]. Cationic polysaccharides can condense large genes into compact structures and neutralize the negative charge of DNA [29]. The main cationic polysaccharides used for gene transfer are natural (such as chitosan) and semi-synthetic derivatives (such as cationic derivatives of cyclodextrin and dextran) [16]. Among them, PEI modified
polysaccharides are representative of semi-synthetic polysaccharide derivatives [30]. Many studies focus on the grafting modification of neutral natural polysaccharides via PEI to obtain safer gene carriers. The results showed that PEI-modified polysaccharides, such as PEI-prulan and PEI-cyclodextrin, had lower toxicity and higher gene transfection efficiency than PEI [30]. Rice bran polysac-
Please cite this article as: L. Liu, Y. Yan, D. Ni et al., TAT-functionalized PEI-grafting rice bran polysaccharides for safe and efficient gene delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.234
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Fig. 8. GFP gene transfection efficiency of PEI30k, PRBP and PRBP-TAT. The cells treated by naked DNA and lipo6000 were set as blank and positive control, respectively, the dosage of GFP plasmid DNA per well was 1 mg or 2 mg. (A) The fluorescence microscope images of cells with positive GFP expression. Scale bar = 500 lm. The cell images were observed by an inverted fluorescence microscope at a magnification of 40. (B) The percentage of cells with positive GFP expression. Results are represented as mean ± SD (n = 3).
Please cite this article as: L. Liu, Y. Yan, D. Ni et al., TAT-functionalized PEI-grafting rice bran polysaccharides for safe and efficient gene delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.234
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charide (RBP), as a cheap substance extracted from rice bran, a byproduct of rice processing, has a wide range of biological activities [31]. In the previous work [19], PRBP was prepared by grafting rice bran polysaccharide RBP with low molecular weight (mw = 600, 2000) PEI and evaluated as gene delivery vector. However, the transfection efficiency of PRBP was unsatisfactory, so PEI30k was recommended to graft RBP in this study. The results showed that PEI30k grafting RBP performed higher gene transfection efficiency than PEI600 and PEI2k grafting RBP, but the cytotoxicity did not increase significantly. When pDNA is mixed with positive charged non-viral vectors, vector/pDNA nanoparticles are formed by DNA condensation, which reduces the molecular size of pDNA [32]. The vecrot/DNA nanoparticles with slightly positive charges can interact electrostatically with cell membranes and thus can be internalized [5]. After entering the cell, these nanoparticles are released through endoplasmic escape and nucleic acid is released into the nucleus for gene expression [8]. Therefore, the efficiency of gene delivery of non-viral vectors is closely related to the efficiency of these processes [5]. In this work, PRBP was functionalized by TAT to improve the gene transfection efficiency via enhancing the cell uptake efficiency of vector/pDNA complex. The results showed that compared with PRBP/pDNA, PRBP-TAT/pDNA plyplexes had higher cell uptake efficiency and gene transfection efficiency (Figs. 7 and 8). This indicates that the strategy of TAT functionalization is effective to improve the transfection efficiency of polysaccharide gene vectors, which is consistent with the relevant reports [23]. At the same time, it is noticed that the prepared PBRP-TAT does not show higher transfection efficiency than commercial reagent lipo6000, which may be due to the ineffective strategy of TAT grafting PRBP, resulting in low grafting ratio. Safety is one of the obstacles that non-viral gene vectors must overcome. Good biosafety is the guarantee of efficient gene delivery and accelerated clinical application of the vector in clinical trials [33]. As Fig. 6 shows, PRBP and PRBP-TAT both exhibit lower cytotoxicity than PEI, which is contributed by the good biocompatibility of RBP. Although TAT grafting resulted in increased cytotoxicity of PRBP, it was not obvious. This suggests that PRBP-TAT has a excellent biocompatibility while performs a satisfactory gene transfection efficiency. In short, although the application of non-viral gene vectors in gene therapy has been extensively studied and made great progress, for some gene delivery, the defects of high toxicity, low transfection efficiency and host response remain to be solved [8]. This work demonstrated that the high molecular weight (Mw = 30,000 Da) PEI-grafted RBP could obtain a higher gene transfection efficiency, and that could be further improved by TAT functionalization. This indicates that PRBP-TAT has potential as a safe and effective gene delivery vector. However, in order to design and develop more effective carriers, further efforts are needed to improve the grafting ratio of TAT to PRBP by optimizing the grafting scheme.
5. Conclusion Rice bran polysaccharide was used as the initial material, and PEI modified rice bran polysaccharide (PRBP) was obtained by grafting rice bran polysaccharide. Then a gene delivery system PRBP-TAT with good biocompatibility and high transfection efficiency was prepared by grafting PRBP with TAT peptide. The results of in vitro evaluation showed that PRBP-TAT could effectively promote DNA condensation and protect DNA from degradation at low N/P ratio, and the efficiency of gene transfection was higher than that of PEI and PRBP. Moreover, PRBP-TAT exhibited lower cytotoxicity to 293T, BRL and HepG2 cells than PEI. These
results are expected to provide a strategy for the development of more efficient polysaccharide-based gene vectors and provide a reference for the application of rice bran polysaccharides as biomaterials. Competing interests The authors declare no conflicts of interest. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (Grant No. 21602166) and the Research and Innovation Initiatives of WHPU (Grant No. 2018Y13). References [1] C.-L. Halioua-Haubold, J.K. Jolly, J.A. Smith, R. Pinedo-Villanueva, D.A. Brindley, R.E. MacLaren, Potential lifetime quality of life benefits of choroideremia gene therapy: projections from a clinically informed decision model, Eye 33 (8) (2019) 1215–1223. [2] C. Manikandan, A. Kaushik, D. Sen, Viral vector: potential therapeutic for glioblastoma multiforme, Can. Gene Ther. (2019). [3] T.B. Lentz, S.J. Gray, R.J. Samulski, Viral vectors for gene delivery to the central nervous system, Neurobiol. Disease 48 (2) (2012) 179–188. [4] H. Xing, M. Lu, T. Yang, H. Liu, Y. Sun, X. Zhao, H. Xu, L. Yang, P. Ding, Structurefunction relationships of nonviral gene vectors: lessons from antimicrobial polymers, Acta Biomater 86 (2019) 15–40. [5] M.A. Mintzer, E.E. Simanek, Nonviral vectors for gene delivery, Chem. Rev. 109 (2) (2009) 259–302. [6] W.F. Lai, W.T. Wong, Design of Polymeric gene carriers for effective intracellular delivery, Trends Biotechnol. 36 (7) (2018) 713–728. [7] S. Taranejoo, R. Chandrasekaran, W. Cheng, K. Hourigan, Bioreducible PEIfunctionalized glycol chitosan: a novel gene vector with reduced cytotoxicity and improved transfection efficiency, Carbohydr. Polym. 153 (2016) 160–168. [8] Y.-P. Xiao, J. Zhang, Y.-H. Liu, J.-H. Zhang, Q.-Y. Yu, Z. Huang, X.-Q. Yu, Low molecular weight PEI-based fluorinated polymers for efficient gene delivery, Eur. J. Med. Chem. 162 (2019) 602–611. [9] S.M. Caroline Diana, M.R. Rekha, Efficacy of vinyl imidazole grafted cationized pullulan and dextran as gene delivery vectors: A comparative study, Int. J. Biol. Macromol. 105 (2017) 947–955. [10] S.R. Pinnapireddy, L. Duse, B. Strehlow, J. Schafer, U. Bakowsky, Composite liposome-PEI/nucleic acid lipopolyplexes for safe and efficient gene delivery and gene knockdown, Colloids Surf B Biointerfaces 158 (2017) 93–101. [11] L. Hasanzadeh, M. Darroudi, N. Ramezanian, P. Zamani, S.H. Aghaee-Bakhtiari, E. Nourmohammadi, R.K. Oskuee, Polyethylenimine-associated cerium oxide nanoparticles: a novel promising gene delivery vector, Life Sci. 116661 (2019). [12] R. Deng, Y. Yue, F. Jin, Y. Chen, H. Kung, M.C.M. Lin, C. Wu, Revisit the complexation of PEI and DNA — How to make low cytotoxic and highly efficient PEI gene transfection non-viral vectors with a controllable chain length and structure?, J. Control. Release 140 (1) (2009) 40–46. [13] D. Sahoo, S. Sahoo, P. Mohanty, S. Sasmal, P.L. Nayak, Chitosan: a new versatile bio-polymer for various applications, Des. Monomers Polym. 12 (5) (2009) 377–404. [14] T. Khan, A. Date, H. Chawda, K. Patel, Polysaccharides as potential anticancer agents – a review of their progress, Carbohydr. Polym. 210 (2019) 412–428. [15] M.J. Amicucci, E. Nandita, C.B. Lebrilla, Function without structures: the need for in-depth analysis of dietary carbohydrates, J. Agric. Food. Chem. 67 (16) (2019) 4418–4424. [16] W. Khan, H. Hosseinkhani, D. Ickowicz, P.-D. Hong, D.-S. Yu, A.J. Domb, Polysaccharide gene transfection agents, Acta Biomaterialia 8 (12) (2012) 4224–4232. [17] L. Wang, Y. Li, L. Zhu, R. Yin, R. Wang, X. Luo, Y. Li, Y. Li, Z. Chen, Antitumor activities and immunomodulatory of rice bran polysaccharides and its sulfates in vitro, Int. J. Biol. Macromol. 88 (2016) 424–432. [18] Y. Rong, R. Yang, Y. Yang, Y. Wen, S. Liu, C. Li, Z. Hu, X. Cheng, W. Li, Structural characterization of an active polysaccharide of longan and evaluation of immunological activity, Carbohydr. Polym. 213 (2019) 247–256. [19] S. Wu, D. Ni, Y. Yan, X. Pan, X. Chen, J. Guan, X. Xiong, L. Liu, Safe and efficient gene delivery based on rice bran polysaccharide, Int. J. Biol. Macromol. 137 (2019) 1041–1049. [20] H. Brooks, B. Lebleu, E. Vives, Tat peptide-mediated cellular delivery: back to basics, Adv. Drug Deliv. Rev. 57 (4) (2005) 559–577. [21] M. Rizzuti, M. Nizzardo, C. Zanetta, A. Ramirez, S. Corti, Therapeutic applications of the cell-penetrating HIV-1 Tat peptide, Drug. Discov. Today 20 (1) (2015) 76–85. [22] V.P. Torchilin, Tat peptide-mediated intracellular delivery of pharmaceutical nanocarriers, Adv. Drug Deliv. Rev. 60 (4–5) (2008) 548–558. [23] M. Malhotra, C. Tomaro-Duchesneau, S. Prakash, Synthesis of TAT peptidetagged PEGylated chitosan nanoparticles for siRNA delivery targeting neurodegenerative diseases, Biomaterials 34 (4) (2013) 1270–1280.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
TAT-functionalized PEI-grafting rice bran polysaccharides for safe and efficient gene delivery Liang Liu a,1,⇑, Yujian Yan a,1, Danni Ni a, Shuheng Wu a, Yiran Chen a, Xin Chen a, Xuemin Xiong a, Gang Liu b,⇑ a b
School of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, China College of Food and Science Engineering, Wuhan Polytechnic University, Wuhan 430023, China
a r t i c l e
i n f o
Article history: Received 12 August 2019 Received in revised form 5 September 2019 Accepted 22 September 2019 Available online xxxx
a b s t r a c t Polysaccharides are considered to be promising candidates for non-viral gene delivery because of their molecular diversity, which can be modified to fine-tune their physicochemical properties. In this work, transcriptional activator protein (TAT) functionalized PEI grafted polysaccharide polymer (PRBP) was prepared by using rice bran polysaccharide as the starting material, and characterized by various methods. The potential of TAT functionalized PRBP (PRBP-TAT) as gene vector was studied in vitro, including DNA loading capacity, DNA protection ability and biocompatibility. The cell uptake and transfection efficiency of the PRBP-TAT/pDNA polyplexes were studied. The results showed that PRBP-TAT could completely condense DNA at N/P 2. The PRBP-TAT/pDNA polyplexes could protect DNA from degrading by DNase and were efficiently internalized by cells. Biocompatibility result showed that PRBP-TAT had no significant cytotoxicity and effect on cell proliferation. At low N/P ratios of 1–3.5, PRBP-TAT showed higher transfection efficiency than PEI30k and PEI30k-grafted rice bran polysaccharide. PRBP-TAT and PEI showed the highest transfection efficiency of 42.8% and 28.1% when pDNA is 2 mg and N/P ratio is 1.5, respectively, while PRBP showed the highest transfection efficiency of 37.3% at N/P 2.5. These results indicate that PTA is a promising candidate vector for safe and efficient gene delivery. Ó 2019 Elsevier B.V. All rights reserved.
1. Introduction As a new cancer treatment strategy, gene therapy has attracted much attention because of its remarkable therapeutic efficacy [1]. However, safe and efficient gene delivery is still one of the main obstacles in gene therapy. Viral gene vectors have been extensively studied due to their efficient transfection efficiency [2]. However, since viral vectors have serious defects including inflammation response, production difficulties, inflammatory reactions, immunogenicity, and carcinogenicity, they are rarely used in actual clinical treatment [3]. Although the gene transfection efficiency of the non-viral vector is lower than that of the viral vector, the toxicity and immunogenicity are lower than the viral vector [4]. Thus, such as liposomes, micelles, cationic polymers, and polyanionic polymers, etc., have been extensively studied to replace viral vectors for efficient gene delivery [5]. Among various non-viral gene carriers, cationic polymers have attracted the attention of researchers due to their unique physicochemical properties [6]. PEI is one of the most widely studied materials as gene carriers [7]. PEI performs high DNA condensation ⇑ Corresponding authors. 1
E-mail addresses: [email protected] (L. Liu), [email protected] (G. Liu). These authors contributed equally to this work.
ability and strong proton sponge effect, which can destroy the endolysosome and internalize the vector/pDNA complex into the cytoplasm, and is therefore considered to be the gold standard for evaluating transfection efficiency [8]. However, as a gene delivery vector, PEI has several problems to be solved: such as severe cytotoxicity of PEI caused by excessive cationic charge [9,10]. Therefore, researchers modified PEI by various biodegradable materials to address this issue [11,12]. Polysaccharides have a variable molecular structure due to the variability of monomers and the stereospecificity of glycosidic linkages and differences in monosaccharide sequences [13]. Polysaccharides have many advantages as biomaterials, such as excellent biocompatibility and biodegradability, which can be obtained from agricultural or marine supplementary food resources [14,15]. Therefore, polysaccharide is widely used to modify PEI to prepare a low toxicity, biodegradable gene and drug vector [16]. Rice bran polysaccharides (RBP) has a wide range of biological activities, including anti-tumor, antioxidant and anti-inflammatory effects, as well as improved diarrhea and immune function [17,18]. So far, RBP is rarely studied as a biological material, especially as drug and gene delivery vector. In previous study, we have synthesized low molecular weight PEI (Mw = 600, 2000 Da) modified RBP (PEI-RBP), and the PEI-RBP exhibited higher transfection efficiency and better biocompatibility than PEI600 and PEI2k. But
https://doi.org/10.1016/j.ijbiomac.2019.09.234 0141-8130/Ó 2019 Elsevier B.V. All rights reserved.
Please cite this article as: L. Liu, Y. Yan, D. Ni et al., TAT-functionalized PEI-grafting rice bran polysaccharides for safe and efficient gene delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.234
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in comparison with commercial transfection reagents of lipo6000, the gene transfection efficiency of PEI-RBP is still unsatisfactory [19]. TAT (transcriptional activator protein) is a short, positively charged peptide that helps macromolecules enter mammalian cells [20]. TAT peptide, containing 11-amino-acid within the HIV-1 TAT protein, is able to act as nuclear localization signal domain and easily translocate both the plasma membrane and the nuclear membrane into the cellular nuclear [21]. Recent studies have shown that many substances conjugated with TAT can penetrate cell membranes and perform their biological functions [22]. TAT has also been used to enhance the efficiency of gene transfection mediated by polysaccharides gene vector [23]. Tang et al. [24] proved that TAT peptide-conjugated hyaluronic acid could condense DNA, and the vector/pDNA polyplexes could be able to efficiently release DNA and deliver DNA into the nuclear, resulted in higher transfection efficiency than PEI/pDNA polyplexes. Yan et al. [25] reported the TAT peptide-conjugated N-succinylchitosan carriers for safe and efficient gene transfection and in vitro. On this basis, we hypothesize that the gene transfer efficiency of PEI-RBP can be further improved by grafting TAT, and a new efficient gene transfer system is expected to be discovered. In this study, in order to develop safe and efficient gene delivery vector based on RBP, PEI30k modified rice bran polysaccharides (PRBP) and TAT conjugated PRBP (PRBP-TAT) were prepared. The structure and chemical composition of PRBP-TAT were analyzed by FTIR, 1H NMR and elemental analysis. DNA binding and protection ability, biocompatibility of PRBP-TAT and DNA release, cell uptake and transfection efficiency of PRBP-TAT/pDNA were studied in vitro. This work is the first effort to improve the efficiency of PRBP-mediated gene transfection through TAT conjugation, and provide insights for the research and development of RBP as a biomaterial. 2. Materials and methods 2.1. Materials Rice bran (RB) was obtained from Hubei Hunsen Biotechnology Co., Ltd (Hubei, China) and the rice bran polysaccharides (RBP) were prepared and determined according to our previous work
[26]. Commercial branched polyethylenimines with average Mw 30 k Da (bPEI30k) was purchased from Macklin Inc (Shanghai, China). TAT (YGRKKRRQRRRC[NH2]) was purchased from Ontores Biotechnology Co., Ltd (Hangzhou, China). Lipo6000 transfection agent was gained from Beyotime Biotechnology (Shanghai, China). PikoGreen dsDNA quantitative Kit was purchased from Shanghai Liji Biotechnology Co., Ltd (Shanghai, China). The EGFP plasmid was amplified in E. coli, then extracted and purified by a plasmid extraction kit (Omega Bio-Tek, Guangzhou, China) according to the manufacturer’s protocol. 3-(4,5-dimethylthiazol-2-yl)-2,5-dip henyltetrazolium bromide (MTT), 4,6-diamidino 2-phenylindole (FITC) wase purchased from Sigma-Aldrich (Shanghai, China). Fetal bovine serum (FBS) and other cell culture medium were purchased from Gibco (New York, USA). Human embryonic kidney cells (293T), rat hepatocytes (BRL) and liver hepatocellular cells (HepG2) were purchased from National infrastructure of cell line resource (Beijing, China). Other biochemical reagents are analytical pure and purchased from Sinopharm Co., Ltd (Shanghai, China). 2.2. Preparation of PRBP and PRBP-TAT PRBP and PRBP-TAT were prepared according to the scheme in Fig. 1. PRBP: 500 mg RBP was reacted with 500 mg CDI in 150 mL DMSO containing 2 mL triethylamine for 3 h at rt, under nitrogen atmosphere, and protected from light. 50 mg PEI30k was then dissolved in 20 mL DMSO and dropped to the reaction mixture. After 24 h, the mixture was centrifuged, and the supernatant was collected and dialyzed in a dialysis bag to remove small molecular impurities. Three times of absolute ethanol was added to the dialysis interception solution, and the mixture was stayed overnight at 4 °C. Then precipitation was collected by centrifugation and pure PRBP was obtained by freeze-drying. The successful preparation of PRBP was confirmed by FTIR (Nicolet Avatar-370, Thermal Fisher Scientific, USA) and 1H NMR (Ascend400, BRUKER, Switzerland). PRBP-TAT: 100 mg PRBP and 100 mg CDI were reacted in 50 mL DMSO containing 2 mL triethylamine in a nitrogen atmosphere for 3 h. 10 mg TAT was dissolved in 5 mL DMSO, and then added to the above reaction solution, and the reaction was continued for 24 h. Dialysis (molecular weight cutoff, 3000 Da) was carried out to remove redundant TAT, and the dialyzate was freeze-dried to
Fig. 1. The preparation scheme of PRBP and PRBP-TAT.
Please cite this article as: L. Liu, Y. Yan, D. Ni et al., TAT-functionalized PEI-grafting rice bran polysaccharides for safe and efficient gene delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.234
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obtain PRBP-TAT. The successful preparation of PRBP-TAT was confirmed by FTIR and 1H NMR.
2.3. Preparation of FITC labeling PRBP and PRBP-TAT Fluorescein isothiocyanate (FITC) labeling PRBP (FITC-PRBP) and PRBP-TAT (FITC-PRBP-TAT) were synthesized to evaluate the cellular uptake efficiency of vector/pDNA polyplexes. 5 mg PRBP and 0.2 mg FITC were reacted in deionized water at rt for 24 h. Then the dialysis assay was used to remove the free FITC, and the FITC-PRBP was obtained by freeze-drying. FITC-PRBP-TAT was prepared in the same method. FITC-PRBP and FITC-PRBP-TAT were characterized by fluorescence spectroscopy (LS55, PerkinElmer, USA) and UV–Vis spectroscopy (Lambda 25, PerkinElmer, USA).
2.4. Characterization The prepared PRBP and PRBP-TAT were characterized by FTIR (Fourier transform infrared spectroscopy), 1H NMR and elemental analysis. For FTIR analysis, 3 mg PRBP or PRBP-TAT was mixed with 600 mg potassium bromide (KBr) in a mortar, and then used to press tablet. A infrared spectrometer (PerkinElmer Frontier, PerkinElmer, USA) was used to measured the absorption at wave number range of 600–4000 cm 1. For 1H NMR, 10 mg PRBP and PRBPTAT was dissolved in 1 mL D2O, respectively, then loaded into a nuclear magnetic resonance tube for analysis (Ascend 400, Bruker, USA). Elemental analysis was carried out on a FLASH 2000 NC analyzer (Thermo Fisher Scientific, USA).
2.5. Preparation of vector/pDNA polyplexes The PRBP-TAT/pDNA polyplexes with various N/P ratios were prepared for different assays. EGFP plasmid DNA and different dosage PRBP-TAT were incubated in DMEM solution at 37 °C for 30 min to obtain the PRBP-TAT/pDNA polyplexes. The polyplexes contain 0.2 lg EGFP pDNA was used to prepare the PRBP-TAT/ pDNA polyplexes for gel retardation assay, and 1 lg for cell uptake and gene transfection assays. The vector/pDNA polyplexes DMEM solution was mixed by vortex before use. The PRBP/pDNA polyplexes were prepared in the same way, but PRBP is used instead of PRBP-TAT. The vector/pDNA polyplexes were dispersed in 0.001 M KCl solution and their particle size and zeta potential were determined by a Zetasizer Nano S90 (Malvern, UK).
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2.7. Cell culture 293T, BRL and HepG2 cells were used in the MTT, RTCA, cell uptake and EGFP gene transfection assays. Cells were cultured in high glucose DMEM supplemented with 10% (v/v) heatinactivated fetal bovine serum (FBS) and 1% antibiotic agent (penicillin and streptomycin) at 37 °C in a 5% CO2 atmosphere. 2.8. MTT and RTCA assay The cytotoxicity of PRBP-TAT was evaluated by MTT assay in 293T, BRL and HepG2 cells. Cells were seeded in 96-well plate at density of 1 104 cells/well and cultured in complete medium for 24 h. Then the culture medium was removed and various concentrations (0.005, 0.01, 0.015, 0.02, 0.025 and 0.03 mg/mL) PRBPTAT were added to cells, respectively. After incubation for 36 h, the medium was removed and the cells were washed by PBS for three times, and then the cells were treated with 10 mL 0.5% (m/v) MTT DMEM solution for 4 h. 150 mL DMSO was then used to replace the MTT solution and dissolve the formazan crystals. The absorbance of each well at 490 nm (A490) was measured by a microplate reader (EnspireTM, PerkinElmer, USA). The cells treated with DMEM were set as blank and six replicates were made for each concentration of the PRBP-TAT. The cytotoxicity of PRBP-TAT/pDNA polyplexes were studied as the same method. PRBP-TAT/pDNA polyplexes were prepared according to Section 2.5, and the N/P ratios were 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5. For comparison, the cytotoxicity of PEI30k, PEI30k/pDNA, PRBP and PRBP/pDNA polyplexes was also studied. RTCA is based on the analysis of cell activity related to the growth trend of adherent cells via an xCELLigence RTCA S16 device (ACEA Biosciences, Inc., Hangzhou, China). RTCA detects the effect of cell adherence on the electric resistance of an e-plate which is consistent with cell viability in real time. RTCA assay was performed according to the manufacturer’s instructions. 200 mL complete medium was added into each well of the e-plate to calibrate the instrument baseline. Then, cells were seeded into each well and cultured in 5% CO2 at 37 °C for 30 min. The e-plate was placed on the detector and culturing continued for 24 h until the cell concentration was approximately 70–80%. After that, the culture medium was removed, complete media containing different concentrations of PRBP-TAT were added into each well, respectively, and the cells were continually cultured in 5% CO2 at 37 °C for 48 h. The cell index data were recorded every 5 min during the beginning 2 h, then per 2 h during the later stage. The data were exported after the end of experiment through a manufacturer’s software of RTCA iCELLigence V2.0.
2.6. DNA release 2.9. Cell uptake assay Vector/DNA polyplexes were prepared according to Section 2.5. The reaction mixture was centrifuged to remove the DMEM and free DNA. Then the vector/DNA polyplexes were dispersed in 5 mL PBS (phosphate buffer solution, pH 7.4) and sodium acetate buffer solution (pH 5.0), respectively, and the solution was shaken at 100 rpm at 37 °C in a biochemical incubator. To study the DNA release, the suspension was centrifuged after various time, then 10 lL supernatant was removed and the amount of DNA was measured by a PikoGreen dsDNA quantitative Kit according to the manufacturer’s instructions. After each measurement, the total volume of suspension was supplemented with DMEM to 5 mL to keep the total volume unchanged. The DNA release ratio was calculated as: DNA release ratio = (m1/m0) 100%. m1 is the amount of DNA in vector/DNA polyplexes, m0 is the amount of releasing DNA from vector/DNA polyplexes. Vector represents PRBP or PRBP-TAT in this section. The standard error of the mean was calculated from the three independent measurements.
Cell uptake is one of the main obstacles in the process of gene delivery by non-viral vectors. Effective cell uptake is the prerequisite for efficient gene transfection. In this work, the uptake efficiency of PRBP-TAT/pDNA and PRBP/pDNA polyplexes by 293T cells were investigated by fluorescence microscope and flow cytometry. In order to realize cell uptake results visualization, FITC labeled PRBP-TAT and PRBP were used to form the vector/pDNA polyplexes. The N/P ratios were 1 to 3.5 for FITC-PRBP/pDNA and FITC-PRBP-TAT/pDNA polyplexes. 293T cells were seeded in 12well plate at a density of 1 105 cells/well and cultured in complete medium until the cell density reached 70–80%. Then the DMEM dispersion of FITC-vector/pDNA polyplexes with various N/P ratios was used to replace the the culture medium, respectively, and the cells were cultured for 4 h. Cells were then fixed with 1% paraformaldehyde and observed by an inverted fluorescence microscope (ix53, Olympus, Japan). Moreover, the cellular
Please cite this article as: L. Liu, Y. Yan, D. Ni et al., TAT-functionalized PEI-grafting rice bran polysaccharides for safe and efficient gene delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.234
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uptake efficiency of FITC-vector/pDNA polyplexes was quantitatively measured by flow cytometer (FACSCanto II, BD, USA). The data are given as mean ± standard deviation (SD) based on 3 measurements. 2.10. In vitro gene transfection study In order to investigate the influence of pDNA doses on transfection efficiency, vector/pDNA polyplexes with the same N/P ratio were formed with EGFP plasmid DNA of 1 and 2 ug, respectively, according to Section 2.5. The N/P ratios were 1 to 3.5 for PRBP/ pDNA, PRBP-TAT/pDNA, and PEI30k/pDNA. Lipo6000 was used as positive control and naked DNA as blank control. For lipo6000 mediated plasmid transfection, 1.0 mg plasmid DNA and 2.0 mL lipo6000 transfection reagent were added in 5 mL DMEM, respectively, then mixed and incubated at room temperature for 10 min before transfection. The ratio of plasmid (mg) to Lipo6000 (mL) was 1:2 (m/v). 293T cells were seeded in 24-well plates with density of 3 l04 cells/well and cultured in complete medium until
reached 70–80% confluence, and then the medium was removed and cells were washed with PBS. The vector/pDNA polyplexes were slowly added to the cells and cultured for 4 h. Then 1 mL complete medium was used to replace the primary medium and cultured for 48 h. The transfection result was observed by fluorescence microscope and quantitatively measured by flow cytometer. For flow cytometer assay, the transfected cells were digested by trypsin and washed by PBS, then dispersed in the working fluid and the positive expression EGFP cells were measured by flow cytometer. The data are given as mean ± standard deviation (SD) based on 3 measurements. 3. Results 3.1. Characterization The 1H NMR spectrum was used to confirm the successful preparation of PRBP and PRBP-TAT. The 1H NMR spectra of RBP (A), PEI30k (B), PRBP (C) and PRBP-TAT (D) are shown in Fig. 2.
(A)
(B)
RBP
PEI30k
(D)
(C) PRBP
PRBP-TAT
(E)
Fig. 2. The 1H NMR spectra of RBP (A), PEI30k (B), PRBP30k (C), PRBP-TAT (D) and the FTIR spectra of RBP, PRBP and PRBP-TAT.
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H NMR usually provides the information of glycoside bond configuration and hydrogen number ratio in polysaccharide structure. However, due to the similar chemical shifts of non-heteropoly H and partial overlap, most H signals are difficult to analyze. The chemical shifts of general b heteropoly region are in the range of d 4.4 – 5.0, and those of a heteropoly region are in the range of d 5.0 – 5.5. The 1H NMR signals of RBP have d 5.209 and 4.613 which indicate that there are two glycoside configurations of a and b in RBP. The peaks at 2.4–2.6 ppm in the NMR spectra of PEI30k, PRBP and PRBP-TAT copolymers correspond to –NHCH2CH2– in PEI, which indicates that PEI is conjugated to RBP. The peak at 1– 2 ppm is attributed to –CH2– of TAT, which appears in the PRBPTAT spectrum, indicating that the TAT peptide was successfully coupled to PRBP. The FTIR spectra of RBP, PRBP and PRBP-TAT are shown in Fig. 2E. For RBP, the peak of 3200–3400 cm 1 is caused by the stretching vibration of –OH, the peaks near 2390 cm 1 are caused by C–H vibration. Compared with RBP, the PRBP FTIR spectra shows that the peak intensity at 3200–3400 cm 1 is decreased, which is due to the reducing of hydroxyl caused by the PEI conjugation. The peak of 1600 cm 1 contributes to vibration of N–H, the peak of 1000–1200 cm 1 contributes to the characteristic peak of C–O–C, the peak of 1300 cm 1 is caused by the stretching vibration of C–N, and peak of 1570 cm 1 is caused by vibration of N–H. These results indicate that the grafting of PEI30k onto RBP is successful. The peak at 1450–1650 cm 1 is the absorption peak of tyrosine benzene ring in TAT peptide. This indicates that the successfully TAT grafting of PRBP. The contents of C, N and H of PRBP and PRBP0-TAT were studied by elemental analysis. As shown in Table 1, the result exhibits that the content of N and C slightly decreases from PRBP to PRBP-TAT, while H increases by 2%. This is because the percentage of N and C in PRBP is higher than that in TAT, while the content of H is lower than that in TAT. The N/P ratios of PRBP-TAT/pDNA and PRBP/ pDNA polyplexes are calculated according to the elemental analysis results. The elemental analysis further proves that TAT is successfully grafted to PRBP.
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3.2. Particle size and zeta potential of vector/pDNA polyplexes To determine the particle size of vector/pDNA polyplexes, pDNA was complexed with PRBP or PRBP-TAT at various N/P ratios of 1– 3.5, then analyzed using DLS. As shown in Fig. 3A, the particle size of vector/pDNA polyplexes were decreasing with the increase of N/P ratio. At N/P 3.5, the PRBP/ pDNA and PRBP-TAT/pDNA polyplexes have a average particle size of less than 200 nm and are suitable for cellular uptake. PRBP-TAT/pDNA polyplexes have a smaller particle size than PRBP/pDNA at different N/P ratios. This is due to the fact that the charge density of PRBP-TAT (Fig. 3B) is higher than PRBP and thus has a stronger DNA condensation ability. This means that the zeta potential of PRBP-TAT benefits from the TAT conjunction. Moreover, the result (Fig. 3B) shows that the zeta potential of various vector/pDNA polyplexes is increased relative to N/P ratios. 3.3. DNA loading and protection Naked DNA can be degraded by DNase I, and enzymatic degradation contributes to a major limitation for gene delivery. Therefore, DNA condensation and protection ability is one of the basic capabilities for non-viral gene vectors. The interactions of RBP based vectors with ctDNA were investigated by agarose gel electrophoresis. As shown in Fig. 4A, the results show that PEI30k, PRBP and PRBP-TAT can condensate DNA at low N/P ratio. The minimum N/P ratio which is needed to condensed DNA is 1.5 for PEI30k, 2.5 for PRBP and 1 for PRBP-TAT. This is because the conjugation of RBP decreases the positive charge density of PEI30k, while the modification of TAT increases the positive charge density of PRBP. The protective ability of PEI30k, PRBP and PRBP-TAT to DNA is shown in Fig. 4B. The results showed that after incubation with DNase I for 4 h, the naked pDNA was completely degraded, but the DNA present in the form of vector/DNA polyplex (which was blocked in the loading well) was not degraded. These results indicate that PRBP-TAT can effectively condense and protect DNA from enzymatic degradation. 3.4. DNA release
Table 1 The contents of C, N, H in PRBP and PRBP-TAT. Element
Content (%) in PRBP
Content (%) in PRBP-TAT
C N H
46.78 24.85 8.10
44.36 23.59 10.10
The DNA release was evaluated by PicoGreen kit. As shown in Fig. 5, the release profile of plasmid from vector/pDNA polyplex displays a biphasic pattern that was characterized by a first rapid release within 2 h, followed by a slower and sustained release within 10 h, at pH 7.4. The release of the plasmid rate of PRBP/ pDNA and was somewhat slower than those of PRBP-TAT/pDNA. The results exhibit that PRBP-TAT/pDNA not only has a higher
Fig. 3. Particle size (A) and zeta potential (B) of vector/pDNA polyplexes.
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(A)
(B)
positively correlated with their charge density. RBP modification reduces the charge density of PEI, while TAT grafting increases the charge density of PRBP. The cytotoxicity of vector/pDNA polyplexes with various N/P ratio was shown as Fig. 6D. It shows that PRBP-TAT/pDAN polyplexes show lower cytotoxicity than PEI30k/ pDNA and PRBP/pDNA. These results suggest that TAT functionalization increases the cytotoxicity of PRBP, but doesn’t effect the PRBP-TAT/pDAN polyplexes exhibit a good biocompatibility. This is due to DNA neutralizing the positive charge density of PRBPTAT in polyplexes (Fig. 3B). RTCA assay was used to analyze the effects of vectors on cell growth on-line. As Fig. 6E and F show, PRBP and PRBP-TAT manifest a slight inhibitory effect on the growth of BRL and HepG2 cells, while PEI30k shows more significant inhibitory effect on cell growth. This is consistent with the results of MTT assay, and it is asserted that PRBP-TAT has a better biocompatibility than PEI30k. 3.6. Cell uptake
Fig. 4. DNA binding (A) and protection (B) ability of PEI30k, PRBP and PRPB-TAT.
Fig. 5. DNA release ratio of PRBP/pDNA and PRBP-TAT/pDNA polyplexes at various pH condition. Results are represented as mean ± SD (n = 3).
DNA release rate, but also a high release ratio at pH 5.0 than PRBP/ pDNA. This suggests that PRBP-TAT/pDNA pilyplexes can release DNA efficiently and may perform more efficient gene transfection than PRBP/pDNA.
The vector/pDNA polyplexes with fluorescent probes were prepared by FITC-labeled vectors. Then the cell absorption of the polyplexes was studied by fluorescence microscopy and flow cytometry, respectively. As shown in Fig. 7A and B, the UV–visible absorption spectrum and fluorescence emission spectrum of FITC labeled PRBP and PRBP-TAT had similar characteristic peaks with FITC, but they were completely different before labeling. The slight red shift in the labeled PBP and PBP-TAT spectra is attributed to the change of pH caused by amino group in PEI. This results indicate that PRBP and PRBP-TAT are successfully labeled by FITC. The DNA condensation ability of FITC labeled PRBP and PRBP-TAT was examined by gel retardation assay. The result shows (Fig. 7C) that FITC-PRBP and FITC-PRBP-TAT can completely condensate DNA at N/P 2 and 1, respectively. Therefore, it is feasible to use FITC to label the vector to study the cellular uptake of the vector/pDNA polyplexes. An inverted fluorescence microscopy was used to observe the cell uptake of the labeled vector/pDNA polyplexes. As shown in Fig. 7D, after 4 h of treatment with various N/P ratios of FITClabeled vectors/pDNA polyplexes, obvious green fluorescence was observed in the cells, and the fluorescence intensity increases with the increase of N/P. This is attributed by the decrease of particle size of polyplexes caused by the increase of N/P (Fig. 3A) [5]. The results manifest that the polyplexes can be efficiently absorbed by cells. Flow cytometry (Fig. 7E) results confirmed that PRBPTAT/pDNA and PRBP/pDNA polyplexes could be internalized in cells, and the uptake efficiency increased with the increase of N/P ratio. It showed that the uptake efficiency of PRBP-TATt/pDNA was 95% at N/P 3.5, while that of PRBP/pDNA was 65%. In the N/P range of 1–3.5, PRBP-TAT/pDNA polyplexes exhibited higher the uptake efficiency than PRBP/pDNA, indicating that the cell uptake efficiency of PRBP was improved by the TAT peptide functionalization. 3.7. In vitro gene transfection study
3.5. Biocompatibility MTT assay was used to evaluate the cytotoxicity of PRBP-TAT and PRBP-TAT/pDNA to HepG2, BRL, and 293T cells. Cells were treated with various concentrations of PRBP-TAT, respectively. The concentration range of PRBP-TAT was consistent with that of uptake and gene transfection assays, ranging from 5 to 30 mg/mL. The cytotoxicity of PEI30k and PRBP was evaluated for comparison. Fig. 6A, B and C show that RBP is not cytotoxic to the three cells, conversely, PEI shows strong cytotoxicity. Different samples show the following sequence of cytotoxicity to cells: PEI > PRBP-TAT > P RBP > RBP. This is because the cytotoxicity of non-viral vectors is
PRBP-TAT-mediated gene transfection was carried out in 293T cells. 1 mg and 2 mg EGFP encoding plasmid DNA were used as the reporter gene, respectively. Cells treated with DMEM were set as blank control and commercial reagent lipo6000 was set as positive control. For comparison, the transfection mediated by PEI30k and PBRP were also evaluated. As Fig. 8A shows, after 24 h of treatment with different vector/pDNA polyplexes with N/ P ratios of 1–3.5, obvious GFP expression (green fluorescence) was observed in cells, but not in cells treated with naked DNA (data not show). Compared with PRBP/pDNA, PRBP-TAT/pDNA showed more significant GFP expression.
Please cite this article as: L. Liu, Y. Yan, D. Ni et al., TAT-functionalized PEI-grafting rice bran polysaccharides for safe and efficient gene delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.234
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Fig. 6. The biocompatibility results. (A) The cell viability of 293T, (B) BRL and (C) HepG2 cells after treated with various concentration of RPB, PEI30k, PRBP and PRBP-TAT. (D) The cell viability of 293T cells after treated with PEI30k/pDNA, PRBP/pDNA and PRBP-TAT/pDNA polyplexes with various of N/P ratios. The real time cell index of BRL (E) and HepG2 (F) cells after treated with various concentration of RPB, PEI30k, PRBP and PRBP-TAT. Results are represented as mean ± SD (n = 6).
Flow cytometry was used to quantitatively determine the transfection efficiency of different vectors. The transfection efficiency increases with the increasing of pDNA dosage, as shown in Fig. 8B, the transfection efficiency of vectors with 2 mg DNA was significantly higher than that with 1 mg. However, the transfection efficiency doesn’t further increase with the increasing of pDNA dosage (data not show). It shows that PRBP has higher transfection efficiency than PEI30k. When pDNA is 2 mg and N/P ratio is 2.5, the highest transfection efficiency of PRBP is 37.3%, while PEI shows the highest transfection efficiency of 28.1% at N/P ratio of 1.5. This may be due to the reduction of cytotoxicity of PEI30k by polysaccharide grafting, thus improving the transfection efficiency. Remarkably, PRBP-TAT exhibits higher transfection efficiency than both PEI30k and PRBP. It is shows that PRBP-TAT performs the highest transfection efficiency of 42.8% when pDNA is 2 mg and N/P ratio is 1.5. In addition, the disparity of transfection efficiency
between PRBP-TAT/pDNA and PRBP/pDNA was more significant when 1 mg pDNA was used for transfection experiments. Comparison of the cell uptake results, it can be inferred that the higher transfection efficiency of PRBP-TAT/pDNA is contributed to the higher cell uptake efficiency. This indicates that it is feasible to improve the transfection efficiency through TAT functionalization. 4. Discussion Polysaccharides are non-toxic, biodegradable and biocompatible materials [13]. They are particularly suitable for blood transfusion and biological applications, because they are water-soluble and can be easily transported to cells in vivo, thus serving as effective carriers for delivering drugs combined with them [27]. In addition, polysaccharides have molecular diversity and can be modified to regulate their physical and chemical properties, so they are con-
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Fig. 7. (A) The UV–Vis absorbance spectrum of FITC-PRPB and FITC-PRBP-TAT. (B) The fluorescence emission spectrum of FITC-PRPB and FITC-PRBP-TAT. (C) DNA condensation ability of FITC-PRPB and FITC-PRBP-TAT. (D) Fluorescence microscope images of 293T cells with positive FITC-PRBP/DNA polyplexes uptake, the cells images were observed by an inverted fluorescence microscope at a magnification of 40. Scale bar = 500 lm. (E) Flow cytometry results of cell uptake efficiency of FITC-PRPB/pDNA and FITC-PRBP-TAT/pDNA polyplexes. Results are represented as mean ± SD, (n = 3).
sidered as a promising non-viral gene vector [28]. Cationic polysaccharides can condense large genes into compact structures and neutralize the negative charge of DNA [29]. The main cationic polysaccharides used for gene transfer are natural (such as chitosan) and semi-synthetic derivatives (such as cationic derivatives of cyclodextrin and dextran) [16]. Among them, PEI modified
polysaccharides are representative of semi-synthetic polysaccharide derivatives [30]. Many studies focus on the grafting modification of neutral natural polysaccharides via PEI to obtain safer gene carriers. The results showed that PEI-modified polysaccharides, such as PEI-prulan and PEI-cyclodextrin, had lower toxicity and higher gene transfection efficiency than PEI [30]. Rice bran polysac-
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Fig. 8. GFP gene transfection efficiency of PEI30k, PRBP and PRBP-TAT. The cells treated by naked DNA and lipo6000 were set as blank and positive control, respectively, the dosage of GFP plasmid DNA per well was 1 mg or 2 mg. (A) The fluorescence microscope images of cells with positive GFP expression. Scale bar = 500 lm. The cell images were observed by an inverted fluorescence microscope at a magnification of 40. (B) The percentage of cells with positive GFP expression. Results are represented as mean ± SD (n = 3).
Please cite this article as: L. Liu, Y. Yan, D. Ni et al., TAT-functionalized PEI-grafting rice bran polysaccharides for safe and efficient gene delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.234
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charide (RBP), as a cheap substance extracted from rice bran, a byproduct of rice processing, has a wide range of biological activities [31]. In the previous work [19], PRBP was prepared by grafting rice bran polysaccharide RBP with low molecular weight (mw = 600, 2000) PEI and evaluated as gene delivery vector. However, the transfection efficiency of PRBP was unsatisfactory, so PEI30k was recommended to graft RBP in this study. The results showed that PEI30k grafting RBP performed higher gene transfection efficiency than PEI600 and PEI2k grafting RBP, but the cytotoxicity did not increase significantly. When pDNA is mixed with positive charged non-viral vectors, vector/pDNA nanoparticles are formed by DNA condensation, which reduces the molecular size of pDNA [32]. The vecrot/DNA nanoparticles with slightly positive charges can interact electrostatically with cell membranes and thus can be internalized [5]. After entering the cell, these nanoparticles are released through endoplasmic escape and nucleic acid is released into the nucleus for gene expression [8]. Therefore, the efficiency of gene delivery of non-viral vectors is closely related to the efficiency of these processes [5]. In this work, PRBP was functionalized by TAT to improve the gene transfection efficiency via enhancing the cell uptake efficiency of vector/pDNA complex. The results showed that compared with PRBP/pDNA, PRBP-TAT/pDNA plyplexes had higher cell uptake efficiency and gene transfection efficiency (Figs. 7 and 8). This indicates that the strategy of TAT functionalization is effective to improve the transfection efficiency of polysaccharide gene vectors, which is consistent with the relevant reports [23]. At the same time, it is noticed that the prepared PBRP-TAT does not show higher transfection efficiency than commercial reagent lipo6000, which may be due to the ineffective strategy of TAT grafting PRBP, resulting in low grafting ratio. Safety is one of the obstacles that non-viral gene vectors must overcome. Good biosafety is the guarantee of efficient gene delivery and accelerated clinical application of the vector in clinical trials [33]. As Fig. 6 shows, PRBP and PRBP-TAT both exhibit lower cytotoxicity than PEI, which is contributed by the good biocompatibility of RBP. Although TAT grafting resulted in increased cytotoxicity of PRBP, it was not obvious. This suggests that PRBP-TAT has a excellent biocompatibility while performs a satisfactory gene transfection efficiency. In short, although the application of non-viral gene vectors in gene therapy has been extensively studied and made great progress, for some gene delivery, the defects of high toxicity, low transfection efficiency and host response remain to be solved [8]. This work demonstrated that the high molecular weight (Mw = 30,000 Da) PEI-grafted RBP could obtain a higher gene transfection efficiency, and that could be further improved by TAT functionalization. This indicates that PRBP-TAT has potential as a safe and effective gene delivery vector. However, in order to design and develop more effective carriers, further efforts are needed to improve the grafting ratio of TAT to PRBP by optimizing the grafting scheme.
5. Conclusion Rice bran polysaccharide was used as the initial material, and PEI modified rice bran polysaccharide (PRBP) was obtained by grafting rice bran polysaccharide. Then a gene delivery system PRBP-TAT with good biocompatibility and high transfection efficiency was prepared by grafting PRBP with TAT peptide. The results of in vitro evaluation showed that PRBP-TAT could effectively promote DNA condensation and protect DNA from degradation at low N/P ratio, and the efficiency of gene transfection was higher than that of PEI and PRBP. Moreover, PRBP-TAT exhibited lower cytotoxicity to 293T, BRL and HepG2 cells than PEI. These
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Please cite this article as: L. Liu, Y. Yan, D. Ni et al., TAT-functionalized PEI-grafting rice bran polysaccharides for safe and efficient gene delivery, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.09.234