STUDY ON GENE TRANSFECTION IN BONE MARROW MESENCHYMAL STEM CELLS MEDIATED BY PLASMID OF BONE MORPHOGENETIC PROTEIN 2 LOADED LIPOPOLYSACCHARIDE-AMINE NANOPOLYMERSOMES_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

GUANYun ¹ , WANGQinmei ² , CHENYing ² ,  TENGWei ^1,3 , HUANGHongzhang ³

1. Department of Prosthodontics, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou Guangdong, 510055, P. R. China;
2. Department of Oral and Maxillofacial Surgery, Affiliated Hospital of Stomatology, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou Guangdong, 510055, P. R. China;
3. Key Laboratory on Assisted Circulation of Ministry of Health, Cardiovascular Division of First Affiliated Hospital, Sun Yat-sen University;

Corresponding author：

TENGWei, Email: tengwei2004@hotmail.com

Keywords：

Gene therapy; Lipopolysaccharide-amine nanopolymersomes; Plasmid of bone morphogenetic protein 2; Bone marrow mesenchymal stem cells; Rat

DOI：

10.7507/1002-1892.20140279

Video：

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Objective To evaluate the combination of lipopolysaccharide-amine nanopolymersomes (LNPs), as a gene vector, with target gene and the transfection in bone marrow mesenchymal stem cells (BMSCs) so as to provide a preliminary experiment basis for combination treatment of bone defect with gene therapy mediated by LNPs and stem cells. Methods Plasmid of bone morphogenetic protein 2 (pBMP-2)-loaded LNPs (pLNPs) were prepared. The binding ability of pLNPs to pBMP-2 was evaluated by a gel retardation experiment with different ratios of nitrogen to phosphorus elements (N/P). The morphology of pLNPs (N/P=60) was observed under transmission electron microscope (TEM) and atomic force microscope (AFM). The size and Zeta potential were measured by dynamic light scattering (DLS). The resistance of pLNPs against DNase I degradation over time was explored. The viability of BMSCs, transfection efficiency, and expression of target protein were investigated after transfection by pLNPs in vitro. Results At N/P≥1.5, pLNPs could completely retard pBMP-2; at N/P of 60, pLNPs was uniform vesicular shape under AFM; TEM observation demonstrated that pLNPs were spherical nano-vesicles with the diameter of (72.07±11.03) nm, DLS observation showed that the size of pLNPs was (123±6) nm and Zeta potential was 20 mV; pLNPs could completely resist DNase I degradation within 4 hours, and such protection capacity to pBMP-2 decreased slightly at 6 hours. The cell survival rate first increased and then decreased with the increase of N/P, and reached the maximum value at N/P of 45; the cytotoxicity was in grade I at N/P≤90, which meant no toxicity for in vivo experiment. While the transfection efficiency of pLNPs increased with the increase of N/P, and reached the maximum value at N/P of 60. So it is comprehensively determined that the best N/P was 60. At 4 days, transfected BMSCs expressed BMP-2 continuously at a relatively high level at N/P of 60. Conclusion LNPs can compress pBMP-2 effectively to form the nanovesicles complex, which protects the target gene against enzymolysis. LNPs has higher transfection efficiency and produces more amount of protein than polyethylenimine 25k and Lipofectamine 2000.

Citation： GUANYun, WANGQinmei, CHENYing, TENGWei, HUANGHongzhang. STUDY ON GENE TRANSFECTION IN BONE MARROW MESENCHYMAL STEM CELLS MEDIATED BY PLASMID OF BONE MORPHOGENETIC PROTEIN 2 LOADED LIPOPOLYSACCHARIDE-AMINE NANOPOLYMERSOMES. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(10): 1292-1297. doi: 10.7507/1002-1892.20140279 Copy

1.	Corsi K, Chellat F, Yahia L, et al.Mesenchymal stem cells, MG63 and HEK293 transfection using chitosan-DNA nanoparticles.Biomaterials, 2003, 24(7):1255-1264.
2.	Evans C.Gene therapy for the regeneration of bone.Injury, 2011, 42(6):599-604.
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6.	Neovius E, Lemberger M, Docherty SA, et al.Alveolar bone healing accompanied by severe swelling in cleft children treated with bone morphogenetic protein-2 delivered by hydrogel.J Plast Reconstr Aesthet Surg, 2013, 66(1):37-42.
7.	Chen G, Deng C, Li YP.TGF-beta and BMP signaling in osteoblast differentiation and bone formation.Int J Biol Sci, 2012, 8(2):272-288.
8.	Luo T, Zhang W, Shi B, et al.Enhanced bone regeneration around dental implant with bone morphogenetic protein 2 gene and vascular endothelial growth factor protein delivery.Clin Oral Implants Res, 2012, 23(4):467-473.
9.	黄忠辉, 滕伟, 陈盈, 等.Alg-g-PEI/pVEGF复合物体内促血管生成作用.生物工程学报, 2013, 29(12):1817-1827.
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12.	Rampersad SN.Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays.Sensors (Basel), 2012, 12(9):12347-12360.
13.	Schoonen WG, Stevenson JC, Westerink WM, et al.Cytotoxic effects of 109 reference compounds on rat H4ⅡE and human HepG2 hepatocytes.Ⅲ:Mechanistic assays on oxygen consumption with MitoXpress and NAD (P) H production with Alamar BlueTM.Toxicol In Vitro, 2012, 26(3):511-525.
14.	Boura JS, Santos FD, Gimble JM, et al.Direct head-to-head comparison of cationic liposome-mediated gene delivery to mesenchymal stem/stromal cells of different human sources:a comprehensive study.Hum Gene Ther Methods, 2013, 24(1):38-48.
15.	Pathak A, Patnaik S, Gupta KC.Recent trends in non-viral vector-mediated gene delivery.Biotechnol J, 2009, 4(11):1559-1572.
16.	Yue Y, Jin F, Deng R, et al.Revisit complexation between DNA and polyethyleneimine-effect of uncomplexed chains free in the solution mixture on gene transfection.J Control Release, 2011, 155(1):67-76.
17.	Pawar SN, Edgar KJ.Alginate derivatization:a review of chemistry, properties and applications.Biomaterials, 2012, 33(11):3279-3305.
18.	He W, Guo Z, Wen Y, et al.Alginate-graft-PEI as a gene delivery vector with high efficiency and low cytotoxicity.J Biomater Sci Polym Ed, 2012, 23(1-4):315-331.
19.	Neamnark A, Suwantong O, Bahadur RK, et al.Aliphatic lipid substitution on 2 kDa polyethylenimine improves plasmid delivery and transgene expression.Mol Pharm, 2009, 6(6):1798-1815.
20.	Xu L, Anchordoquy TJ.Effect of cholesterol nanodomains on the targeting of lipid-based gene delivery in cultured cells.Mol Pharm, 2010, 7(4):1311-1317.
21.	Hsu CY, Hendzel M, Uluda H.Improved transfection efficiency of an aliphatic lipid substituted 2 kDa polyethylenimine is attributed to enhanced nuclear association and uptake in rat bone marrow stromal cell.J Gene Med, 2011, 13(1):46-59.

1. Corsi K, Chellat F, Yahia L, et al.Mesenchymal stem cells, MG63 and HEK293 transfection using chitosan-DNA nanoparticles.Biomaterials, 2003, 24(7):1255-1264.
2. Evans C.Gene therapy for the regeneration of bone.Injury, 2011, 42(6):599-604.
3. Huang Z, Teng W, Liu L, et al.Efficient cytosolic delivery mediated by polymersomes facilely prepared from a degradable, amphiphilic, and amphoteric copolymer.Nanotechnology, 2013, 24(26):265104.
4. Rastegar F, Shenaq D, Huang J, et al.Mesenchymal stem cells:Molecular characteristics and clinical applications.World J Stem Cells, 2010, 2(4):67-80.
5. Ben-David D, Srouji S, Shapira-Schweitzer K, et al.Low dose BMP-2 treatment for bone repair using a PEGylated fibrinogen hydrogel matrix.Biomaterials, 2013, 34(12):2902-2910.
6. Neovius E, Lemberger M, Docherty SA, et al.Alveolar bone healing accompanied by severe swelling in cleft children treated with bone morphogenetic protein-2 delivered by hydrogel.J Plast Reconstr Aesthet Surg, 2013, 66(1):37-42.
7. Chen G, Deng C, Li YP.TGF-beta and BMP signaling in osteoblast differentiation and bone formation.Int J Biol Sci, 2012, 8(2):272-288.
8. Luo T, Zhang W, Shi B, et al.Enhanced bone regeneration around dental implant with bone morphogenetic protein 2 gene and vascular endothelial growth factor protein delivery.Clin Oral Implants Res, 2012, 23(4):467-473.
9. 黄忠辉, 滕伟, 陈盈, 等.Alg-g-PEI/pVEGF复合物体内促血管生成作用.生物工程学报, 2013, 29(12):1817-1827.
10. 舒晓春, 朱丹华, 刘君静, 等.大鼠骨髓间充质干细胞体外生长增殖特点的实验研究.中华预防医学杂志, 2010, 44(10):923-927.
11. 刘健康, 杨亚利.大鼠骨髓间充质干细胞的分离、培养及表型鉴定.国际外科学杂志, 2011, 38(10):674-676.
12. Rampersad SN.Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays.Sensors (Basel), 2012, 12(9):12347-12360.
13. Schoonen WG, Stevenson JC, Westerink WM, et al.Cytotoxic effects of 109 reference compounds on rat H4ⅡE and human HepG2 hepatocytes.Ⅲ:Mechanistic assays on oxygen consumption with MitoXpress and NAD (P) H production with Alamar BlueTM.Toxicol In Vitro, 2012, 26(3):511-525.
14. Boura JS, Santos FD, Gimble JM, et al.Direct head-to-head comparison of cationic liposome-mediated gene delivery to mesenchymal stem/stromal cells of different human sources:a comprehensive study.Hum Gene Ther Methods, 2013, 24(1):38-48.
15. Pathak A, Patnaik S, Gupta KC.Recent trends in non-viral vector-mediated gene delivery.Biotechnol J, 2009, 4(11):1559-1572.
16. Yue Y, Jin F, Deng R, et al.Revisit complexation between DNA and polyethyleneimine-effect of uncomplexed chains free in the solution mixture on gene transfection.J Control Release, 2011, 155(1):67-76.
17. Pawar SN, Edgar KJ.Alginate derivatization:a review of chemistry, properties and applications.Biomaterials, 2012, 33(11):3279-3305.
18. He W, Guo Z, Wen Y, et al.Alginate-graft-PEI as a gene delivery vector with high efficiency and low cytotoxicity.J Biomater Sci Polym Ed, 2012, 23(1-4):315-331.
19. Neamnark A, Suwantong O, Bahadur RK, et al.Aliphatic lipid substitution on 2 kDa polyethylenimine improves plasmid delivery and transgene expression.Mol Pharm, 2009, 6(6):1798-1815.
20. Xu L, Anchordoquy TJ.Effect of cholesterol nanodomains on the targeting of lipid-based gene delivery in cultured cells.Mol Pharm, 2010, 7(4):1311-1317.
21. Hsu CY, Hendzel M, Uluda H.Improved transfection efficiency of an aliphatic lipid substituted 2 kDa polyethylenimine is attributed to enhanced nuclear association and uptake in rat bone marrow stromal cell.J Gene Med, 2011, 13(1):46-59.

Chinese Journal of Reparative and Reconstructive Surgery

STUDY ON GENE TRANSFECTION IN BONE MARROW MESENCHYMAL STEM CELLS MEDIATED BY PLASMID OF BONE MORPHOGENETIC PROTEIN 2 LOADED LIPOPOLYSACCHARIDE-AMINE NANOPOLYMERSOMES

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