Enhancement of gene transfection efficiency and therapeutic effect of ultrasound-targeted microbubble destruction <i>in vivo</i> with cationic microbubble _Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANG Dongyang ^1,2 , YANG Lei ¹ , TIAN Hai ¹ , FU Bicheng ¹ , CHEN Wei ¹ , LIU Kaiyu ¹ , JIA Zhibo ¹ , JIANG Shulin ¹ , HAN Xinhao ³ ,  SUN Lu ¹

1. Department of Cardiovascular Surgery, the Second Affiliated Hospital of Harbin Medical University, Harbin Heilongjiang, 150086, P.R.China;
2. Department of Thoracic Surgery, the Affiliated Hospital of Qingdao University, Qingdao Shandong, 266003, P.R.China;
3. Department of Health Supervision, School of Public Health of Harbin Medical University, Harbin Heilongjiang, 150086, P.R.China;

Corresponding author：

SUN Lu, Email: sunlu19802003@163.com

Keywords：

Cationic microbubble; gene transfection; gene therapy; ultrasound-targeted microbubble destruction; ischemic myocardium; rat

DOI：

10.7507/1002-1892.201706058

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ObjectiveTo construct a cationic microbubble (CMB), and investigate the enhancement of gene transfection efficiency and therapeutic effect of ultrasound-targeted microbubble destruction (UTMD) in vivo with CMB compared to definity MB (DMB).Methods In vitro, the CMB was prepared by the method of thin film hydration. The morphology, size, zeta potential, and gene-carrying capacity of CMB were compared with the DMB. In vivo, the firefly luciferase gene which was used as a reporter gene was targeted transfected into myocardium of 16 rats with CMB and DMB, respectively. The gene transfection efficiency and targeting were observed dynamically. Then, ischemia-reperfusion (I/R) model was performed on 64 rats. The models of 60 rats were successfully confirmed by using ultrasonography at 5 days after I/R. The rats were divided into 3 groups (n=20) randomly. The control group received DMB carrying empty plasmid for transfection; DMB group received DMB carrying AKT plasmid for transfection; and CMB group received CMB carrying AKT plasmid for transfection. The cardiac perfusion, cardiac function, infarct size, and infarct thickness were measured by ultrasonography and histological observations after treatment. In addition, the capillary and arteriolar densities were measured with immunohistochemical staining. The myocyte apoptosis was measured with TUNEL staining. The protein expressions of AKT, phospho-AKT (P-AKT), Survivin, and phospho-BAD (P-BAD) were measured by Western blot.ResultsThe size of CMB was uniformly. The zeta potential of CMB was significantly higher than that of DMB (t=28.680, P=0.000). The CMB bound more plasmid DNA than the DMB (P<0.05). The luciferase activity of myocardium were higher in CMB group than in DMB group bothin vitro and in vivo measurements (P<0.05). There was no significant difference between groups in the ratio of signal intensity in anterior wall to posterior wall, ejection fraction (EF), and fractional shortening (FS) at 5 days after I/R (P>0.05), but the above indexes were significant higher in CMB and DMB groups than in control group at 21 days after I/R (P<0.05). Besides, the above indexes were significant higher in CMB group than in DMB group at 21 days after I/R (P<0.05). The infarct size was the smallest and infarct thickness was the thickest in the CMB group, followed by DMB group, control group at 21 days after I/R. The capillary and arteriolar densities of CMB and DMB groups were significant higher than those of control group at 21 days after I/R (P<0.05). Besides, the capillary and arteriolar densities of CMB group were significant higher than those of DMB group (P<0.05). The apoptotic cells were the most in the control group, followed by DMB group, CMB group at 3 days after gene transfection, showing significant differences between groups (P<0.05). The protein expressions of AKT, P-AKT, Survivin, and P-BAD were significant higher in CMB and DMB groups than those in control group at 3 days after gene transfection (P<0.05). Besides, these protein expressions were significant higher in CMB group than those in DMB group (P<0.05).ConclusionThe DNA-carrying capacity and gene transfection efficiency are elevated by CMB, although its physicochemical property is the same as DMB. When ultrasound-targeted AKT gene transfection is used to treat myocardial I/R injury in rats, delivery of AKT with the CMB can result in higher transfection efficiency and greater cardiac functional improvements compared to the DMB.

Citation： ZHANG Dongyang, YANG Lei, TIAN Hai, FU Bicheng, CHEN Wei, LIU Kaiyu, JIA Zhibo, JIANG Shulin, HAN Xinhao, SUN Lu. Enhancement of gene transfection efficiency and therapeutic effect of ultrasound-targeted microbubble destruction in vivo with cationic microbubble . Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(2): 228-236. doi: 10.7507/1002-1892.201706058 Copy

1.	St John Sutton M, Pfeffer MA, Moye L, et al. Cardiovascular death and left ventricular remodeling two years after myocardial infarction: baseline predictors and impact of long-term use of captopril: information from the Survival and Ventricular Enlargement (SAVE) trial. Circulation, 1997, 96(10): 3294-3299.
2.	Sutton MG, Sharpe N. Left ventricular remodeling after myocardial infarction: pathophysiology and therapy. Circulation, 2000, 101(25): 2981-2988.
3.	Bekeredjian R, Grayburn PA, Shohet RV. Use of ultrasound contrast agents for gene or drug delivery in cardiovascular medicine. J Am Coll Cardiol, 2005, 45(3): 329-335.
4.	Yau TM, Fung K, Weisel RD, et al. Enhanced myocardial angiogenesis by gene transfer with transplanted cells. Circulation, 2001, 104(12 Suppl 1): I218-222.
5.	Chen S, Shohet RV, Bekeredjian R, et al. Optimization of ultrasound parameters for cardiac gene delivery of adenoviral or plasmid deoxyribonucleic acid by Ultrasound-targeted microbubble destruction. J Am Coll Cardiol, 2003, 42(2): 301-308.
6.	Fujii H, Sun Z, Li SH, et al. Ultrasound-targeted gene delivery induces angiogenesis after a myocardial infarction in mice. JACC Cardiovasc Imaging, 2009, 2(7): 869-879.
7.	Fujii H, Li SH, Wu J, et al. Repeated and targeted transfer of angiogenic plasmids into the infarcted rat heart via ultrasound targeted microbubble destruction enhances cardiac repair. Eur Heart J, 2011, 32(16): 2075-2084.
8.	Zhao YZ, Lu CT, Li XK, et al. Improving the cardio protective effect of aFGF in ischemic myocardium with ultrasound-mediated cavitation of heparin modified microbubbles: preliminary experiment. J Drug Target, 2012, 20(7): 623-631.
9.	Song S, Shen Z, Chen L, et al. Explorations of high-intensity therapeutic ultrasound and microbubble-mediated gene delivery in mouse liver. Gene Ther, 2011, 18(10): 1006-1014.
10.	Smith DA, Porter TM, Martinez J, et al. Destruction thresholds of echogenic liposomes with clinical diagnostic ultrasound. Ultrasound Med Biol, 2007, 33(5): 797-809.
11.	Fedak PWM, Altamentova SM, Weisel RD, et al. Matrix remodeling in experimental and human heart failure: a possible regulatory role for TIMP-3. Am J Physiol Heart Circ Physiol, 2003, 284(2): H626-634.
12.	Song G, Ouyang G, Bao S. The activation of Akt/PKB signaling pathway and cell survival. J Cell Mol Med, 2005, 9(1): 59-71.
13.	Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three Akts. Genes Dev, 1999, 13(22): 2905-2927.
14.	Mangi AA, Noiseux N, Kong D, et al. Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts. Nat Med, 2003, 9(9): 1195.
15.	Shiojima I, Walsh K. Role of Akt signaling in vascular homeostasis and angiogenesis. Circ Res, 2002, 90(12): 1243-1250.
16.	Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell, 1997, 91(2): 231-241.
17.	Li F, Ambrosini G, Chu EY, et al. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature, 1998, 396(6711): 580-584.
18.	Lentacker I, De Geest BG, Vandenbroucke RE, et al. Ultrasound-responsive polymer-coated microbubbles that bind and protect DNA. Langmuir, 2006, 22(17): 7273-7278.
19.	Liu F, Shollenberger LM, Conwell CC, et al. Mechanism of naked DNA clearance after intravenous injection. J Gene Med, 2007, 9(7): 613-619.
20.	Panje CM, Wang DS, Pysz MA, et al. Ultrasound-mediated gene delivery with cationic versus neutral microbubbles: effect of DNA and microbubble dose on in vivo transfection efficiency. Theranostics, 2012, 2(11): 1078-1091.
21.	Houk BE, Hochhaus G, Hughes JA. Kinetic modeling of plasmid DNA degradation in rat plasma. AAPS PharmSci, 1999, 1(3): 15-20.

1. St John Sutton M, Pfeffer MA, Moye L, et al. Cardiovascular death and left ventricular remodeling two years after myocardial infarction: baseline predictors and impact of long-term use of captopril: information from the Survival and Ventricular Enlargement (SAVE) trial. Circulation, 1997, 96(10): 3294-3299.
2. Sutton MG, Sharpe N. Left ventricular remodeling after myocardial infarction: pathophysiology and therapy. Circulation, 2000, 101(25): 2981-2988.
3. Bekeredjian R, Grayburn PA, Shohet RV. Use of ultrasound contrast agents for gene or drug delivery in cardiovascular medicine. J Am Coll Cardiol, 2005, 45(3): 329-335.
4. Yau TM, Fung K, Weisel RD, et al. Enhanced myocardial angiogenesis by gene transfer with transplanted cells. Circulation, 2001, 104(12 Suppl 1): I218-222.
5. Chen S, Shohet RV, Bekeredjian R, et al. Optimization of ultrasound parameters for cardiac gene delivery of adenoviral or plasmid deoxyribonucleic acid by Ultrasound-targeted microbubble destruction. J Am Coll Cardiol, 2003, 42(2): 301-308.
6. Fujii H, Sun Z, Li SH, et al. Ultrasound-targeted gene delivery induces angiogenesis after a myocardial infarction in mice. JACC Cardiovasc Imaging, 2009, 2(7): 869-879.
7. Fujii H, Li SH, Wu J, et al. Repeated and targeted transfer of angiogenic plasmids into the infarcted rat heart via ultrasound targeted microbubble destruction enhances cardiac repair. Eur Heart J, 2011, 32(16): 2075-2084.
8. Zhao YZ, Lu CT, Li XK, et al. Improving the cardio protective effect of aFGF in ischemic myocardium with ultrasound-mediated cavitation of heparin modified microbubbles: preliminary experiment. J Drug Target, 2012, 20(7): 623-631.
9. Song S, Shen Z, Chen L, et al. Explorations of high-intensity therapeutic ultrasound and microbubble-mediated gene delivery in mouse liver. Gene Ther, 2011, 18(10): 1006-1014.
10. Smith DA, Porter TM, Martinez J, et al. Destruction thresholds of echogenic liposomes with clinical diagnostic ultrasound. Ultrasound Med Biol, 2007, 33(5): 797-809.
11. Fedak PWM, Altamentova SM, Weisel RD, et al. Matrix remodeling in experimental and human heart failure: a possible regulatory role for TIMP-3. Am J Physiol Heart Circ Physiol, 2003, 284(2): H626-634.
12. Song G, Ouyang G, Bao S. The activation of Akt/PKB signaling pathway and cell survival. J Cell Mol Med, 2005, 9(1): 59-71.
13. Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three Akts. Genes Dev, 1999, 13(22): 2905-2927.
14. Mangi AA, Noiseux N, Kong D, et al. Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts. Nat Med, 2003, 9(9): 1195.
15. Shiojima I, Walsh K. Role of Akt signaling in vascular homeostasis and angiogenesis. Circ Res, 2002, 90(12): 1243-1250.
16. Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell, 1997, 91(2): 231-241.
17. Li F, Ambrosini G, Chu EY, et al. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature, 1998, 396(6711): 580-584.
18. Lentacker I, De Geest BG, Vandenbroucke RE, et al. Ultrasound-responsive polymer-coated microbubbles that bind and protect DNA. Langmuir, 2006, 22(17): 7273-7278.
19. Liu F, Shollenberger LM, Conwell CC, et al. Mechanism of naked DNA clearance after intravenous injection. J Gene Med, 2007, 9(7): 613-619.
20. Panje CM, Wang DS, Pysz MA, et al. Ultrasound-mediated gene delivery with cationic versus neutral microbubbles: effect of DNA and microbubble dose on in vivo transfection efficiency. Theranostics, 2012, 2(11): 1078-1091.
21. Houk BE, Hochhaus G, Hughes JA. Kinetic modeling of plasmid DNA degradation in rat plasma. AAPS PharmSci, 1999, 1(3): 15-20.

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