Journal of Materials Chemistry B
Paper
transfection efficiencies in 10% FBS medium were close to those
in serum-free medium. Fig. 8b–j show the scatter diagrams of
transfection in the three cells measured by ow cytometry. In
the diagrams, the dots of the Q4 region (the lower right)
represent the number of cells expressing green uorescence
protein (GFP), and the dots of Q3 region (the lower le) repre-
sent the number of cells not expressing GFP. Fig. 8b–d showed
that in the three blank samples only very few dots in the Q4
region were found, suggesting that almost no transfection
occurred. In Fig. 8e–j, the percentages of the dots in the Q4
region to dots in (Q3 + Q4) regions should be equal to the values
of the transfection efficiencies in Fig. 8a.
2 K. Ma, M. X. Hu, Y. Qi, J. H. Zou, L. Y. Qiu, Y. Jin, X. Y. Ying
and H. Y. Sun, Biomaterials, 2009, 30, 6109–6118.
3 H. M. Wu, S. R. Pan, M. W. Chen, Y. Wu, C. Wang, Y. T. Wen,
X. Zeng and C. B. Wu, Biomaterials, 2011, 32, 1619–
1634.
4 Y. T. Wen, Z. H. Guo, Z. Du, R. Fang, H. M. Wu, X. Zeng,
C. Wang, M. Feng and S. R. Pan, Biomaterials, 2012, 33,
8111–8121.
5 Y. Wang, W. Kong, Y. Song, Y. Duan, L. Wang, G. Steinhoff,
D. Kong and Y. Yu, Biomacromolecules, 2009, 10, 617–622.
6 G. Navarro and C. Tros de ILarduya, Nanomed.: Nanotechnol.,
Biol. Med., 2009, 5, 287–297.
To quantify the serum tolerance, i.e. serum-resistant capacity
for transfection, we measured the rates of transfection effi-
ciency of polymer/pDNA complexes at a xed weight ratio in
7 X. Q. Zhang, X. L. Wang, S. W. Huang, R. X. Zhuo, Z. L. Liu,
H. Q. Mao and K. W. Leong, Biomacromolecules, 2005, 6, 341–
350.
1
0% FBS medium to that in serum-free medium (abbreviated
8 Y. Shoji and H. Nakashima, Curr. Pharm. Des., 2004, 10, 785–
796.
9 N. Shah, R. J. Steptoe and H. S. Parekh, J. Pept. Sci., 2011, 17,
470–478.
as: the rates of transfection efficiency with serum to without
serum). As shown in Table 5, for EA-G2 at w/w ¼ 50 in Hek-293,
Cos-7 and Bel-7402 cells, the rates were 1.09, 1.04 or 0.94. For
PEI-25k at N/P ¼ 10 in the three cells, the rates were only 0.33, 10 W. Tansey, S. Ke, X. Y. Cao, M. J. Pasuelo, S. Wallace and
.14 or 0.08. For EA-G1 at w/w ¼ 50 in Hek-293 cells and Lipo-2k C. Li, J. Controlled Release, 2004, 94, 39–51.
complexes at w/w ¼ 8 in Hek-293 cells, the rates were 0.48 or 11 J. Khandare, A. Mohr, M. Calder ´o n, P. Welker, K. Licha and
.28, respectively. The above results indicated that EA-G2 R. Haag, Biomaterials, 2010, 31, 4268–4277.
complexes displayed greater serum tolerance than PEI-25k, EA- 12 L. Chen, H. Tian, J. Chen, X. Chen, Y. Huang and X. Jing, J.
0
0
G1 and Lipo-2k complexes.
Gene Med., 2010, 12, 64–76.
13 S. K. Tripathi, R. Goyal, K. M. Ansari, K. Ravi Ram, Y. Shukla,
D. K. Chowdhuri and K. C. Gupta, Eur. J. Pharm. Biopharm.,
4
Conclusion
2
011, 79, 473–484.
A novel PEG-PLGA copolymer (EA-G2) was prepared by an ami- 14 Y. T. Wen, S. R. Pan, X. Luo, X. Zhang, W. Zhang and
nolysis of PEG-PBLG copolymer using PAMAM G2. The analysis M. Feng, Bioconjugate Chem., 2009, 20, 322–332.
of FT-IR, H-NMR, DSC and GPC conrmed the formation of EA- 15 X. Zeng, S. R. Pan, J. Li, C. Wang, Y. T. Wen, H. M. Wu,
1
G2. The EA-G2 exhibited considerable biodegradability, low
cytotoxicity and great ability to combine with pDNA. The EA-G2/
C. F. Wang, C. B. Wu and M. Feng, Nanotechnology, 2011,
22, 375102.
pDNA complexes showed particle sizes in the range of 120–180 16 S. R. Pan, C. Wang, X. Zeng, Y. T. Wen, H. M. Wu and
nm and the zeta potentials in the range of 20–40 mV. Cellular M. Feng, Int. J. Pharm., 2011, 420, 206–215.
uptake of the EA-G2 complexes occurred mainly through clathrin- 17 A. Harada and K. Kataoka, Macromolecules, 2003, 36, 4995–
dependent and caveolin-mediated endocytosis. In 10% FBS and 5001.
serum-free media, the percentages of the complex uptake at 6 h 18 S. R. Pan, Q. M. Wang and W. Yi, J. Biomater. Appl., 2007, 22,
reached 89.0 or 72.7%, respectively. EA-G2 could efficiently 181–192.
mediate the plasmid EGFP-Cl into the cell nucleus. EA-G2 19 T. Kurosaki, T. Morishita, Y. Kodama, K. Sato, H. Nakagawa,
complexes displayed enhanced transfection efficiency and much
better serum tolerance. EA-G2 has potential to be used as a
biodegradable, efficient and serum-resistant gene vector.
N. Higuchi, T. Nakamura, T. Hamamoto, H. Sasaki and
T. Kitahara, Mol. Pharmaceutics, 2011, 8, 913–919.
20 D. Y. Olton, J. M. Close, C. S. Sfeir and P. N. Kumta,
Biomaterials, 2011, 32, 7662–7670.
2
1 Y. Shen, H. Peng, S. R. Pan, M. Feng, Y. T. Wen, J. J. Deng,
X. Luo and C. B. Wu, Nanotechnology, 2010, 21, 045102.
Acknowledgements
The authors are thankful to the National Natural Science 22 H. Huang, C. M. Dong and Y. Wei, Comb. Chem. High
Foundation of China (30870618 and 31170918) for nancial Throughput Screening, 2007, 10, 368–376.
support on the projects, and to the Key Laboratory on Assisted 23 R. Qi, Y. Gao, Y. Tang, R. R. He, T. L. Liu, Y. He, S. Sun,
Circulation, Ministry of Health for the cooperation in chemical
preparation, cell culture, gene transfection test and so on.
B. Y. Li, Y. B. Li and G. Liu, AAPS J., 2009, 11, 395–405.
24 N. S. Murthy, W. Wang and J. Kohn, Polymer, 2010, 51, 3978–
3988.
2
5 G. Z. Rong, M. X. Deng, C. Deng, Z. H. Tang, L. H. Piao,
X. S. Chen and X. B. Jing, Biomacromolecules, 2003, 4,
1800–1804.
References
1
T. Kurosaki, Y. Yamashita, K. Aki, H. Harasawa, H. Nakagawa,
Y. Kodama, N. Higuchi, T. Nakamura, T. Kitahara and 26 J. Mijovic, S. Ristic and J. Kenny, Macromolecules, 2007, 40,
H. Sasaki, J. Pharm. Sci., 2011, 100, 4855–4863. 5212–5221.
5
126 | J. Mater. Chem. B, 2013, 1, 5114–5127
This journal is ª The Royal Society of Chemistry 2013