Preparation of polyethylenimine-chitosan/DNA nanoparticles for transfecting articular chondrocytes in vitro

doi:10.3969/j.issn.2095-4344.2013.47.004

Abstract

Abstract:

BACKGROUND: Chitosan is well known as good biocompatibility and biodegradability; however, its extensive use in biomedical applications is restricted due to its poor transfection efficiency.
OBJECTIVE: To prepare the polyethyleneimine-chitosan/DNA nanoparticles loading enhanced green fluorescent protein gene, and to detect their physicochemical properties and gene transfection efficiency towards chondrocytes in vitro.
METHODS: Low molecular weight polyethyleneimine was covalently linked to chitosan backbone to construct chitosan-graft-polyethyleneimine; then the chitosan-graft-polyethyleneimine was mixed with DNA nanoparticles, which loaded enhanced green fluorescent protein gene, by a complex coacervation method. The nanoparticle morphology was observed under a scanning electron microscopy. The sizes and zeta-potentials of the nanoparticles were measured by a Marven-nano laser diffractometer. The binding capacity of plasmid DNA was evaluated by agarose gel electrophoresis analysis. The gene transfection experiments in vitro were performed towards rabbit’s chondrocytes. The gene transfection efficiency was measured with flow cytometry and under fluorescence microscope. How marked DNA entered into the nucleus of chondrocytes mediated by the nanoparticles was detected by laser scanning confocal microscopy.
RESULTS AND CONCLUSION: The prepared nanoparticles were mainly spherical, with an average size of (154.6±18.6) nm, and zeta-potential of (24.68±6.82) mV. The agarose gel electrophoresis analysis confirmed that the nanoparticles could effectively protect plasmid DNA from DNase Ⅰ-induced degradation. Gene transfection in vitro proved that the nanoparticles were efficient in transfecting rabbit’s chondrocytes and the expression of green fluorescent proteins was observed under fluorescent microscope, with a transfection efficiency of (23.80±1.74)% that was significantly higher than that of the naked plasmid DNA and chitosan/DNA nanoparticles (P < 0.05). But no significant differences were observed between polyethyleneimine-chitosan/DNA nanoparticles and LipofectamineTM 2000. These findings indicate that the polyethyleneimine-chitosan/DNA nanoparticles can effectively protect plasmid DNA from nuclease degradation, and exhibit the favorable transfection ability towards articular chondrocytes.

Key words: biocompatible materials, chitosan, polyethyleneimine, chondrocytes

CLC Number:

R318

Lu Hua-ding, Dai Yu-hu, Lian Li-yi, Lü Lu-lu, Zhao Hui-qing. Preparation of polyethylenimine-chitosan/DNA nanoparticles for transfecting articular chondrocytes in vitro[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(47): 8162-8168.

Figures/Tables 5

References

[1] Rekha MR,Sharma CP.Polymers for gene delivery: current status and future perspectives.Recent Pat DNA Gene Seq. 2012;6(2):98-107.
[2] Tiera MJ,Shi Q,Winnik FM,et al.Polycation-based gene therapy: current knowledge and new perspectives.Curr Gene Ther.2011;11(4):288-306.
[3] Tripathi SK,Goyal R,Kumar P,et al.Linear polyethylenimine-graft-chitosan copolymers as efficient DNA/siRNA delivery vectors in vitro and in vivo. Nanomedicine.2012;8(3):337-345.
[4] Mao S,Sun W,Kissel T.Chitosan-based formulations for delivery of DNA and siRNA.Adv Drug Deliv Rev.2010; 62(1):12-27.
[5] Gao JQ,Zhao QQ,Lv TF,et al.Gene-carried chitosan-linked-PEI induced high gene transfection efficiency with low toxicity and significant tumor-suppressive activity.Int J Pharm.2010;387(1-2):286-294.
[6] 卢华定,赵慧清,吕璐璐,等.壳聚糖-负载增强型绿色荧光蛋白基因的质粒DNA纳米微球体外转染软骨细胞的能力及影响因素[J].中华创伤骨科杂志,2011,13(11):64-69．
[7] Steinert AF,Nöth U,Tuan RS.Concepts in gene therapy for cartilage repair. Injury.2008;39 Suppl 1:S97-113.
[8] Lehrman S.Virus treatment questioned after gene therapy death. Nature.1999;401(6753):517-518.
[9] Liu Q,Muruve DA.Molecular basis of the inflammatory response to adenovirus vectors.Gene Ther.2003;10(11): 935-940.
[10] 张世浩,朱立新,靳安民,等.软骨细胞复合胶原/壳聚糖/β-磷酸三钙层状梯度修复体的体外培养[J].中国组织工程研究与临床康复,2008,12(41):8033-8036．
[11] Jayakumar R,Chennazhi KP,Muzzarelli RAA,et al.Chitosan conjugated DNA nanoparticles in gene therapy.Carbohydrate Polymers.2010;79(1):1-8.
[12] Aiba S.Studies on chitosan: 2. Solution stability and reactivity of partially N-acetylated chitosan derivatives in aqueous media. Int J Biol Macromol.1989;11(4): 249-252.
[13] Wong K,Sun G,Zhang X,et al.PEI-g-chitosan, a novel gene delivery system with transfection efficiency comparable to polyethylenimine in vitro and after liver administration in vivo. Bioconjug Chem.2006;17(1): 152-158.
[14] Lavertu M,Méthot S,Tran-Khanh N,et al. High efficiency gene transfer using chitosan/DNA nanoparticles with specificcombinationsof molecular weight and degree of deacetylation. Biomaterials.2006;27(27):4815-4824.
[15] Chang KL,Higuchi Y,Kawakami S,et al. Efficient gene transfection by histidine-modified chitosan through enhancement ofendosomal escape.Bioconjug Chem. 2010; 21(6):1087-1095.
[16] Song B,Zhang W,Peng R,et al.Synthesis and cell activity of novel galactosylated chitosan as a gene carrier.Colloids Surf B Biointerfaces. 2009;70(2):181-186.
[17] Xu Z,Wan X,Zhang W,et al.Synthesis of biodegradable polycationic methoxy poly (ethylene glycol)– polyethylenimine–chitosan and its potential as gene carrier. Carbohydr Polym.2009;78:46-53.
[18] Jiang HL,Kim YK,Arote R,et al.Chitosan-graft- polyethylenimine as a gene carrier.J Control Release.2007; 117(2):273-280.
[19] Gao Y,Xu Z,Chen S,et al. Arginine-chitosan/DNA self-assemble nanoparticles for gene delivery: In vitro characteristics and transfection efficiency.Int J Pharm.2008; 359(1-2):241-246.
[20] Germershaus O,Mao S,Sitterberg J,et al.Gene delivery using chitosan, trimethyl chitosan or polyethylenglycol-graft- trimethyl chitosan block copolymers: establishment of structure-activity relationships in vitro.J Control Release.2008; 125(2):145-154.
[21] Morris VB,Neethu S,Abraham TE,et al.Studies on the condensation of depolymerized chitosans with DNA for preparing chitosan-DNA nanoparticles for gene delivery applications.J Biomed Mater Res B Appl Biomater.2009;89(2): 282-292.
[22] 陈斌,马世坤,高娴.巯基化壳聚糖-质粒 DNA 纳米粒的制备及相关性质的研究[J]. 天津医科大学学报,2008,14(4):466-469．
[23] Knudson W,Chow G,Knudson CB.CD44-mediated uptake and degradation of hyaluronan.Matrix Biol.2002;21(1):15-23.
[24] Campo GM,Avenoso A,Campo S,et al.Small hyaluronan oligosaccharides induce inflammation by engaging both toll-like-4 and CD44 receptors in human chondrocytes.Biochem Pharmacol.2010;80(4):480-490.
[25] Lu HD,Zhao HQ,Wang K,et al.Novel hyaluronic acid-chitosan nanoparticles as non-viral gene delivery vectors targeting osteoarthritis.Int J Pharm.2011; 420(2):358-365.
[26] Sezer AD,Akbu?a J.Comparison on in vitro characterization of fucospheres and chitosan microspheres encapsulated plasmid DNA (pGM-CSF): formulation design and release characteristics.AAPS Pharm Sci Tech.2009;10(4):1193-1199.
[27] 金才益,王斌,曾忠友,等.壳聚糖基因转移载体在关节软骨细胞中表达的定量分析[J].中国组织工程研究与临床康复,2007,11(45): 9035-9038．
[28] Hashimoto M,Morimoto M,Saimoto H,et al.Lactosylated chitosan for DNA delivery into hepatocytes: the effect of lactosylation on the physicochemical properties and intracellular trafficking of pDNA/chitosan complexes.Bioconjug Chem.2006;17(2):309-316.
[29] Feng M,Lee D,Li P.Intracellular uptake and release of poly(ethyleneimine)-co-poly(methyl methacrylate) nanoparticle/pDNA complexes for gene delivery.Int J Pharm. 2006;311(1-2):209-214.
[30] 卢华定,吕璐璐,赵慧清,等.转化生长因子β1基因缓释的壳聚糖纳米粒制备及体外检测[J].中国组织工程研究,2012,16(12): 2120-2124．