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This article can be cited before page numbers have been issued, to do this please use: N. Shao, T. Dai, Y. Liu and Y. Cheng, Chem. Commun., 2015, DOI: 10.1039/C5CC02300A.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
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A Supramolecular Approach to Improve the Gene Transfection Efficacy of Dendrimers Naimin Shao,a Tianjiao Dai,a Yan Liua and Yiyun Cheng* a
Received 00th January 2012, Accepted 00th January 2012 DOI: 10.1039/x0xx00000x www.rsc.org/
Cyanuric acid is able to form complementary hydrogen bonds with melamine. Here, the specific recognition between cyanuric acid and melamine is used to significantly improve the gene transfection efficacy of low generation dendrimer via a supramolecular approach. Cationic dendrimers are widely used as non-viral gene vectors.1 They have several unique features in gene delivery, e.g. well-defined structure, good monodispersity, and ease of surface modification.2 However, researchers in this field are always puzzled by the dilemma between transfection efficacy and cytotoxicity of cationic dendrimers.3 For example, low generation dendrimers have minimal toxicity but poor transfection efficacy. This is because their polyplexes with nucleic acid can be easily destabilized by anionic proteins abundant in cell culture medium and blood.4 Although use of high molecular weight dendrimers can make the polyplexes more stable and achieve relatively high transfection efficacy, this results in increased cytotoxicity as excess positive charges on the polyplexes can disrupt the cell membranes. To break up the correlation between transfection efficacy and toxicity, low generation dendrimers were cross-linked into nanoclusters using biodegradable linkers for efficient transfection.3a The formed nanoclusters can degrade into small polymers during gene delivery, thus have minimal toxicity on the transfected cells. Alternatively, low generation dendrimers were grafted on biocompatible polymers or nanoparticles to “temporarily” improve the dendrimer size for efficient and safe gene delivery.5 In this study, we report a facile supramolecular strategy to develop efficient and low cytotoxic gene vectors based on low generation dendrimers. Cyanuric acid (CyA) is able to form complementary hydrogen bonding network with melamine or 2,4-diamino-1,3,5triazine (DAT). This specific hydrogen bond recognition is usually used to construct three-dimensional noncovalent assemblies.6 Here, DAT was modified on a generation 3 (G3) polyamidoamine (PAMAM) dendrimer according to the method shown in Figure S1.7 An average number of 13 DAT moieties were conjugated on each G3 dendrimer. The product is termed G3-DAT13. The molecular
This journal is © The Royal Society of Chemistry 2012
structure and NMR characterization of G3-DAT13 are shown in Figure 1a and Figure S1. CyA was added into the G3-DAT13/DNA polyplex to form a stable supramolecular complex for efficient gene delivery via the specific hydrogen bond recognition (Scheme 1).
Scheme 1 The complementary hydrogen bond between CyA and DAT tailors the nanostructure of G3-DAT/DNA polyplex.
The existence of hydrogen bond between CyA and G3-DAT13 is proved by 1H NMR (Figure 1b). The hydrogen bond interactions between CyA and G3-DAT13 induce the downshift of DAT protons (HA, HA’ and HB) in deuterated dimethyl sulphoxide (d6-DMSO). Moreover, the addition of CyA into G3-DAT13 significantly broadens of peaks of G3 dendrimer, suggesting the formation of more stable polyplexes (Figure 1c). At higher temperatures, the peaks of G3 dendrimer in the G3-DAT13/CyA complex (the molar ratio CyA to DAT is 8) are gradually recovered (Figure 1d). This phenomenon can be explained by the breakage of hydrogen bonds at high temperatures, which causes the dis-assembly of G3-DAT13/CyA complex. The addition of CyA significantly decreases the fluorescence intensity emitted by ethidium bromide (EB) in the G3DAT13/DNA polyplex solution (Figure 2a). CyA also enhances DNA complexation ability of G3-DAT13 (Figure S2). Furthermore, the formed G3-DAT13/DNA/CyA complex is more resistant to a polyanionic competitor (heparin) than the G3-DAT13/DNA polyplex (Figure 2b). The hydrogen bond recognition between CyA and DAT causes the polyplex morphology transition from a relatively loose
J. Name., 2012, 00, 1-3 | 1
ChemComm Accepted Manuscript
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Figure 2 (a) EB displacement assay of the G3-DAT13/DNA complexes in the presence of CyA, N/P = 64. (b) Stability of G3-DAT13/DNA/CyA and G3-DAT13/DNA polyplexes in the presence of different heparin concentrations analyzed by agarose gel electrophoresis. TEM images of G3-DAT13/DNA (c) and G3-DAT13/DNA/CyA (d) polyplexes. (e) Size and zeta-potential of G3-DAT13/DNA polyplexes in the presence of CyA analyzed by dynamic light scattering (PDI < 0.3). The N/P ratio of the polyplexes in (b-e) is 64. The molar ratio of CyA to DAT in (b) and (d) is 4. Error bars in (a) and (e) represent the s.e. (n = 3).
Figure 1 (a) Chemical structures of G3-DAT13 conjugates with proton assignments. (b) 1H NMR spectra of G3-DAT13 in the presence of CyA in d6-DMSO at 25 oC. The molar ratios of CyA to DAT are 0, 1, 2, 4 and 8, respectively. (c) 1H NMR spectra of G3-DAT13 in the presence of CyA in D2O at 25 oC. The molar ratios of CyA to DAT are 0, 1, 2, 4 and 8, respectively. (d) 1H NMR spectra of G3-DAT13/CyA complex in D2O at different temperatures (25, 35, 45, 55 and 65 oC, respectively). The molar ratio of CyA to DAT in the complex is 8.
We further investigated the transfection efficacy of G3-DAT13 in the absence and presence of CyA on commonly used cell lines. As shown in Figure 3a-3c and Figure S3, the EGFP transfection efficacy of G3-DAT13 at an N/P ratio of 64 (N represents the number of surface primary amine groups. P represents the number of phosphate anions in the DNA chain. The two amine groups on DAT rings were
2 | J. Name., 2012, 00, 1-3
not included because they are not protonated at pH 7.4, pKa~5.1) on HEK293 cells is less than 1%, however, the EGFP expression efficacy is significantly improved in the presence of CyA (>20%). Efficacy of the G3-DAT13/DNA/CyA complex is much higher than that of unmodified G3 PAMAM dendrimer (N/P = 64) and comparable to that of branched poly(ethylenimine) (PEI) with a molecular weight of 25 kDa (bPEI 25KD). Since the CyA was dissolved in a TE buffer (Tris-EDTA buffer, 50 mM Tris-HCl, 10 mM EDTA, pH 8.0) before addition to the polyplex solution, we also excluded the possibility of TE buffer on improving the transfection efficacy (Figure 3c). Similarly, the addition of CyA improves the luciferase expression efficacy of G3-DAT13 by two orders of magnitude on the same cell line (Figure 3d). The efficacy improvement by CyA and DAT recognition is further confirmed on COS-7 cells (Figure S4). To prove that the hydrogen bond recognition is essential in the transfection efficacy improvement, we synthesized an analogue material of G3-DAT13 as a control. 4,6dimethoxy-1,3,5-triazine (DMT) has a similar molecular size to DAT, but is unable to form complementary hydrogen bond with CyA. An average number of 15 DMT molecules were conjugated on each G3 dendrimer (G3-DMT15). As shown in Figure S5, CyA fails to improve the transfection efficacy of G3-DMT15, suggesting the essential role of complementary hydrogen bond recognition in the transfection efficacy improvement. Diffusion NMR results also reveal that no assembled structures are formed between G3-DMT15 and CyA (Figure S6). It is worth noting that pre-incubation of G3DAT13 with CyA also shows much improved transfection efficacy than untreated G3-DAT13 (Figure S7). The addition of CyA into G3DAT13 does not bring additional toxicity. As shown in Figure S8, no obvious cytotoxicity is observed on the transfected cells when different amounts of CyA are added to G3-DAT13.
This journal is © The Royal Society of Chemistry 2012
ChemComm Accepted Manuscript
Published on 07 May 2015. Downloaded by Freie Universitaet Berlin on 13/05/2015 13:47:49.
nanostructure (Figure 2c) to a solid one (Figure 2d). These results clearly demonstrate that CyA improves the stability of G3DAT13/DNA polyplex. The G3-DAT13/DNA polyplex size is slightly increased after the addition of CyA and the G3-DAT13/DNA/CyA complex has a minimum size when the molar ratio of CyA to DAT is 4 (290 nm, Figure 2e).
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COMMUNICATION DOI: 10.1039/C5CC02300A in the design of efficient and low cytotoxic vectors for non-viral gene delivery. This work was financially supported by the National Natural Science Foundation of China (No. 21322405 and No. 21474030) and the Shanghai Municipal Science and Technology Commission (13QA1401500 and 148014518).
Figure 3 EGFP expressions in HEK293 cells mediated by G3-DAT13 (a) and G3-DAT13/CyA (b) for 48 h. Quantitative analysis of EGFP (c) and luciferase (d) expressions in HEK293 cells mediated by G3-DAT13, G3DAT13/CyA, unmodified G3, G3-DAT13/TE buffer, and bPEI 25KD. The N/P ratio of G3-DAT13/DNA polyplex is 64 and the molar ratio of CyA to DAT is 4. For G3/DNA polyplex, the N/P ratio is 64 and CyA/DAT molar ratio is 4. The optimal N/P ratio of bPEI 25KD is 8. Error bars in (c) and (d) represent the s.e. (n = 3).
To further explore the mechanism of CyA on improving the transfection efficacy, the cellular uptake of G3-DAT13/DNA polyplex in the absence and presence of CyA is measured on HEK293 (Figure 4) and COS-7 (Figure S9) cells. CyA is able to significantly increase cellular uptake of the G3-DAT13/DNA polyplex. The more stable structure of the G3-DAT13/DNA/CyA complex may contribute to this enhanced cellular uptake.
500
G3-DAT13
1
2
∗
G3-DAT13 + 4xCyA 400 300
3
∗∗
200 ∗∗
100 ∗∗
0
0.5
1
2
4
4 5
Time (h)
Figure 4 Cellular uptake of YOYO-1 labeled G3-DAT13/DNA polyplex in the absence or presence of CyA. The N/P ratio of the polyplex is 64 and the molar ratio of CyA to DAT is 4. Error bars represent the s.e. (n = 3). *p < 0.05 and **p < 0.01 by students’ t-test.
Conclusions 6
In summary, we present a facile method to improve transfection efficacy of low generation dendrimers through specific hydrogen bond recognition. The formation of complementary hydrogen bond between DAT and CyA improves the polyplex stability, cellular uptake and transfection efficacy of G3-DAT13. In addition, the addition of CyA molecules does not bring additional toxicity on the transfected cells. The supramolecular approach described in this study provides an innovative concept
This journal is © The Royal Society of Chemistry 2012
7
(a) M. Wang, H. Liu, L. Li and Y. Cheng, Nat. Commun., 2014, 5; (b) C. Dufès, I. F. Uchegbu and A. G. Schätzlein, Adv. Drug Deliv. Rev., 2005, 57, 2177; (c) D. W. Pack, A. S. Hoffman, S. Pun and P. S. Stayton, Nat. Rev. Drug Discov., 2005, 4, 581; (d) X. Liu, J. Zhou, T. Yu, C. Chen, Q. Cheng, K. Sengupta, Y. Huang, H. Li, C. Liu, Y. Wang, P. Posocco, M. Wang, Q. Cui, S. Giorgio, M. Fermeglia, F. Qu, S. Pricl, Y. Shi, Z. Liang, P. Rocchi, J. J. Rossi and L. Peng, Angew. Chem. Int. Ed., 2014, 53, 11822; (e) T. Yu, X. Liu, A. L. Bolcato-Bellemin, Y. Wang, C. Liu, P. Erbacher, F. Qu, P. Rocchi, J. P. Behr and L. Peng, Angew. Chem. Int. Ed., 2012, 51, 8478; (f) H. Liu, Y. Wang, M. Wang, J. Xiao and Y. Cheng, Biomaterials, 2014, 35, 5407. (a) D. A. Tomalia, Prog. Polym. Sci., 2005, 30, 294; (b) R. M. Kannan, E. Nance, S. Kannan and D. A. Tomalia, J. Intern. Med., 2014, 276, 579; (c) A. R. Menjoge, R. M. Kannan and D. A. Tomalia, Drug Discov. Today, 2010, 15, 171; (d) K. Luo, B. He, Y. Wu, Y. Shen and Z. Gu, Biotechnol. Adv., 2014, 32, 818; (e) X. Liu, C. Liu, C. V. Catapano, L. Peng, J. Zhou and P. Rocchi, Biotechnol. Adv., 2014, 32, 844. (a) H. Liu, H. Wang, W. Yang and Y. Cheng, J. Am. Chem. Soc., 2012, 134, 17680; (b) M. Breunig, U. Lungwitz, R. Liebl and A. Goepferich, PNAS, 2007, 104, 14454. E. Mastrobattista and W. E. Hennink, Nat. Mater., 2012, 11, 10. (a) X. Xu, Y. Jian, Y. Li, X. Zhang, Z. Tu and Z. Gu, ACS Nano, 2014, 8, 9255; (b) A. M. Chen, O. Taratula, D. Wei, H. I. Yen, T. Thomas, T. Thomas, T. Minko and H. He, ACS Nano, 2010, 4, 3679; (c) A. J. Kim, N. J. Boylan, J. S. Suk, M. Hwangbo, T. Yu, B. S. Schuster, L. Cebotaru, W. G. Lesniak, J. S. Oh and P. Adstamongkonkul, Angew. Chem. Int. Ed., 2013, 52, 3985; (d) H. Huang, D. Cao, L. Qin, S. Tian, Y. Liang, S. Pan and M. Feng, Mol. Pharm., 2014, 11, 2323. (a) B. J. Cafferty, I. Gállego, M. C. Chen, K. I. Farley, R. Eritja and N. V. Hud, J. Am. Chem. Soc., 2013, 135, 2447; (b) M. C. Chen, B. J. Cafferty, I. Mamajanov, I. Gállego, J. Khanam, R. Krishnamurthy and N. V. Hud, J. Am. Chem. Soc., 2013, 136, 5640; (c) E. Elacqua, D. S. Lye and M. Weck, Acc. Chem. Res., 2014, 47, 2405. N. Shao, H. Wang, B. He, Y. Wang, J. Xiao, Y. Wang, Q. Zhang, Y. Li and Y. Cheng, Biomater. Sci., 2015, 3, 500.
J. Name., 2012, 00, 1-3 | 3
ChemComm Accepted Manuscript
a Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai, 200241, P.R. China. *E-mail: [email protected] Electronic Supplementary Information (ESI) available: Experimental Section, supplemental figures and compound spectra. See DOI: 10.1039/c000000x/
Mean Fluorescence Intensity
Published on 07 May 2015. Downloaded by Freie Universitaet Berlin on 13/05/2015 13:47:49.
Notes and references
ChemComm Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: N. Shao, T. Dai, Y. Liu and Y. Cheng, Chem. Commun., 2015, DOI: 10.1039/C5CC02300A.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
www.rsc.org/chemcomm
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DOI: 10.1039/C5CC02300A
Journal Name
RSCPublishing
Cite this: DOI: 10.1039/x0xx00000x
A Supramolecular Approach to Improve the Gene Transfection Efficacy of Dendrimers Naimin Shao,a Tianjiao Dai,a Yan Liua and Yiyun Cheng* a
Received 00th January 2012, Accepted 00th January 2012 DOI: 10.1039/x0xx00000x www.rsc.org/
Cyanuric acid is able to form complementary hydrogen bonds with melamine. Here, the specific recognition between cyanuric acid and melamine is used to significantly improve the gene transfection efficacy of low generation dendrimer via a supramolecular approach. Cationic dendrimers are widely used as non-viral gene vectors.1 They have several unique features in gene delivery, e.g. well-defined structure, good monodispersity, and ease of surface modification.2 However, researchers in this field are always puzzled by the dilemma between transfection efficacy and cytotoxicity of cationic dendrimers.3 For example, low generation dendrimers have minimal toxicity but poor transfection efficacy. This is because their polyplexes with nucleic acid can be easily destabilized by anionic proteins abundant in cell culture medium and blood.4 Although use of high molecular weight dendrimers can make the polyplexes more stable and achieve relatively high transfection efficacy, this results in increased cytotoxicity as excess positive charges on the polyplexes can disrupt the cell membranes. To break up the correlation between transfection efficacy and toxicity, low generation dendrimers were cross-linked into nanoclusters using biodegradable linkers for efficient transfection.3a The formed nanoclusters can degrade into small polymers during gene delivery, thus have minimal toxicity on the transfected cells. Alternatively, low generation dendrimers were grafted on biocompatible polymers or nanoparticles to “temporarily” improve the dendrimer size for efficient and safe gene delivery.5 In this study, we report a facile supramolecular strategy to develop efficient and low cytotoxic gene vectors based on low generation dendrimers. Cyanuric acid (CyA) is able to form complementary hydrogen bonding network with melamine or 2,4-diamino-1,3,5triazine (DAT). This specific hydrogen bond recognition is usually used to construct three-dimensional noncovalent assemblies.6 Here, DAT was modified on a generation 3 (G3) polyamidoamine (PAMAM) dendrimer according to the method shown in Figure S1.7 An average number of 13 DAT moieties were conjugated on each G3 dendrimer. The product is termed G3-DAT13. The molecular
This journal is © The Royal Society of Chemistry 2012
structure and NMR characterization of G3-DAT13 are shown in Figure 1a and Figure S1. CyA was added into the G3-DAT13/DNA polyplex to form a stable supramolecular complex for efficient gene delivery via the specific hydrogen bond recognition (Scheme 1).
Scheme 1 The complementary hydrogen bond between CyA and DAT tailors the nanostructure of G3-DAT/DNA polyplex.
The existence of hydrogen bond between CyA and G3-DAT13 is proved by 1H NMR (Figure 1b). The hydrogen bond interactions between CyA and G3-DAT13 induce the downshift of DAT protons (HA, HA’ and HB) in deuterated dimethyl sulphoxide (d6-DMSO). Moreover, the addition of CyA into G3-DAT13 significantly broadens of peaks of G3 dendrimer, suggesting the formation of more stable polyplexes (Figure 1c). At higher temperatures, the peaks of G3 dendrimer in the G3-DAT13/CyA complex (the molar ratio CyA to DAT is 8) are gradually recovered (Figure 1d). This phenomenon can be explained by the breakage of hydrogen bonds at high temperatures, which causes the dis-assembly of G3-DAT13/CyA complex. The addition of CyA significantly decreases the fluorescence intensity emitted by ethidium bromide (EB) in the G3DAT13/DNA polyplex solution (Figure 2a). CyA also enhances DNA complexation ability of G3-DAT13 (Figure S2). Furthermore, the formed G3-DAT13/DNA/CyA complex is more resistant to a polyanionic competitor (heparin) than the G3-DAT13/DNA polyplex (Figure 2b). The hydrogen bond recognition between CyA and DAT causes the polyplex morphology transition from a relatively loose
J. Name., 2012, 00, 1-3 | 1
ChemComm Accepted Manuscript
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Figure 2 (a) EB displacement assay of the G3-DAT13/DNA complexes in the presence of CyA, N/P = 64. (b) Stability of G3-DAT13/DNA/CyA and G3-DAT13/DNA polyplexes in the presence of different heparin concentrations analyzed by agarose gel electrophoresis. TEM images of G3-DAT13/DNA (c) and G3-DAT13/DNA/CyA (d) polyplexes. (e) Size and zeta-potential of G3-DAT13/DNA polyplexes in the presence of CyA analyzed by dynamic light scattering (PDI < 0.3). The N/P ratio of the polyplexes in (b-e) is 64. The molar ratio of CyA to DAT in (b) and (d) is 4. Error bars in (a) and (e) represent the s.e. (n = 3).
Figure 1 (a) Chemical structures of G3-DAT13 conjugates with proton assignments. (b) 1H NMR spectra of G3-DAT13 in the presence of CyA in d6-DMSO at 25 oC. The molar ratios of CyA to DAT are 0, 1, 2, 4 and 8, respectively. (c) 1H NMR spectra of G3-DAT13 in the presence of CyA in D2O at 25 oC. The molar ratios of CyA to DAT are 0, 1, 2, 4 and 8, respectively. (d) 1H NMR spectra of G3-DAT13/CyA complex in D2O at different temperatures (25, 35, 45, 55 and 65 oC, respectively). The molar ratio of CyA to DAT in the complex is 8.
We further investigated the transfection efficacy of G3-DAT13 in the absence and presence of CyA on commonly used cell lines. As shown in Figure 3a-3c and Figure S3, the EGFP transfection efficacy of G3-DAT13 at an N/P ratio of 64 (N represents the number of surface primary amine groups. P represents the number of phosphate anions in the DNA chain. The two amine groups on DAT rings were
2 | J. Name., 2012, 00, 1-3
not included because they are not protonated at pH 7.4, pKa~5.1) on HEK293 cells is less than 1%, however, the EGFP expression efficacy is significantly improved in the presence of CyA (>20%). Efficacy of the G3-DAT13/DNA/CyA complex is much higher than that of unmodified G3 PAMAM dendrimer (N/P = 64) and comparable to that of branched poly(ethylenimine) (PEI) with a molecular weight of 25 kDa (bPEI 25KD). Since the CyA was dissolved in a TE buffer (Tris-EDTA buffer, 50 mM Tris-HCl, 10 mM EDTA, pH 8.0) before addition to the polyplex solution, we also excluded the possibility of TE buffer on improving the transfection efficacy (Figure 3c). Similarly, the addition of CyA improves the luciferase expression efficacy of G3-DAT13 by two orders of magnitude on the same cell line (Figure 3d). The efficacy improvement by CyA and DAT recognition is further confirmed on COS-7 cells (Figure S4). To prove that the hydrogen bond recognition is essential in the transfection efficacy improvement, we synthesized an analogue material of G3-DAT13 as a control. 4,6dimethoxy-1,3,5-triazine (DMT) has a similar molecular size to DAT, but is unable to form complementary hydrogen bond with CyA. An average number of 15 DMT molecules were conjugated on each G3 dendrimer (G3-DMT15). As shown in Figure S5, CyA fails to improve the transfection efficacy of G3-DMT15, suggesting the essential role of complementary hydrogen bond recognition in the transfection efficacy improvement. Diffusion NMR results also reveal that no assembled structures are formed between G3-DMT15 and CyA (Figure S6). It is worth noting that pre-incubation of G3DAT13 with CyA also shows much improved transfection efficacy than untreated G3-DAT13 (Figure S7). The addition of CyA into G3DAT13 does not bring additional toxicity. As shown in Figure S8, no obvious cytotoxicity is observed on the transfected cells when different amounts of CyA are added to G3-DAT13.
This journal is © The Royal Society of Chemistry 2012
ChemComm Accepted Manuscript
Published on 07 May 2015. Downloaded by Freie Universitaet Berlin on 13/05/2015 13:47:49.
nanostructure (Figure 2c) to a solid one (Figure 2d). These results clearly demonstrate that CyA improves the stability of G3DAT13/DNA polyplex. The G3-DAT13/DNA polyplex size is slightly increased after the addition of CyA and the G3-DAT13/DNA/CyA complex has a minimum size when the molar ratio of CyA to DAT is 4 (290 nm, Figure 2e).
Page 3 of 3
ChemComm View Article Online
Journal Name
COMMUNICATION DOI: 10.1039/C5CC02300A in the design of efficient and low cytotoxic vectors for non-viral gene delivery. This work was financially supported by the National Natural Science Foundation of China (No. 21322405 and No. 21474030) and the Shanghai Municipal Science and Technology Commission (13QA1401500 and 148014518).
Figure 3 EGFP expressions in HEK293 cells mediated by G3-DAT13 (a) and G3-DAT13/CyA (b) for 48 h. Quantitative analysis of EGFP (c) and luciferase (d) expressions in HEK293 cells mediated by G3-DAT13, G3DAT13/CyA, unmodified G3, G3-DAT13/TE buffer, and bPEI 25KD. The N/P ratio of G3-DAT13/DNA polyplex is 64 and the molar ratio of CyA to DAT is 4. For G3/DNA polyplex, the N/P ratio is 64 and CyA/DAT molar ratio is 4. The optimal N/P ratio of bPEI 25KD is 8. Error bars in (c) and (d) represent the s.e. (n = 3).
To further explore the mechanism of CyA on improving the transfection efficacy, the cellular uptake of G3-DAT13/DNA polyplex in the absence and presence of CyA is measured on HEK293 (Figure 4) and COS-7 (Figure S9) cells. CyA is able to significantly increase cellular uptake of the G3-DAT13/DNA polyplex. The more stable structure of the G3-DAT13/DNA/CyA complex may contribute to this enhanced cellular uptake.
500
G3-DAT13
1
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∗
G3-DAT13 + 4xCyA 400 300
3
∗∗
200 ∗∗
100 ∗∗
0
0.5
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4 5
Time (h)
Figure 4 Cellular uptake of YOYO-1 labeled G3-DAT13/DNA polyplex in the absence or presence of CyA. The N/P ratio of the polyplex is 64 and the molar ratio of CyA to DAT is 4. Error bars represent the s.e. (n = 3). *p < 0.05 and **p < 0.01 by students’ t-test.
Conclusions 6
In summary, we present a facile method to improve transfection efficacy of low generation dendrimers through specific hydrogen bond recognition. The formation of complementary hydrogen bond between DAT and CyA improves the polyplex stability, cellular uptake and transfection efficacy of G3-DAT13. In addition, the addition of CyA molecules does not bring additional toxicity on the transfected cells. The supramolecular approach described in this study provides an innovative concept
This journal is © The Royal Society of Chemistry 2012
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ChemComm Accepted Manuscript
a Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai, 200241, P.R. China. *E-mail: [email protected] Electronic Supplementary Information (ESI) available: Experimental Section, supplemental figures and compound spectra. See DOI: 10.1039/c000000x/
Mean Fluorescence Intensity
Published on 07 May 2015. Downloaded by Freie Universitaet Berlin on 13/05/2015 13:47:49.
Notes and references
