树状大分子包裹的纳米金颗粒载体人铁蛋白重链转染自然杀伤细胞92和磁共振成像
|
摘要
目的以聚乙二醇化树状大分子包裹的纳米金颗粒作为非病毒载体,将人铁蛋白重链(FTH1)转染到自然杀伤细胞92(NK92细胞)中,并进行体外细胞MRI检查。方法将第5代聚酰胺-胺树状大分子用聚乙二醇(PEG)修饰之后包裹纳米金颗粒作为非病毒载体,压缩FTH1报告基因然后转染到NK92细胞内。应用磁共振氢谱、紫外分光光度计和透射电子显微镜等对合成的载体材料进行表征;采用琼脂糖凝胶电泳实验确定载体材料与相应基因之间最佳压缩的氮磷比;应用细胞计数试剂盒-8(CCK-8)检测载体材料的毒性;通过测定水动力粒径来确定不同氮磷比的条件下载体材料和基因形成的复合物能否顺利通过细胞膜;应用荧光显微镜和流式细胞仪测定转染效率;应用3.0T的MRI对转染FTH1的NK92细胞进行体外成像。结果合成的聚乙二醇化的包裹纳米金颗粒的树状大分子{(Au~0)_(25)-G5.NH_2-mPEG_(17)}DENPs具有较好的生物相容性,材料细胞毒性较小,在浓度达到3 000nmol/L时细胞存活率仍然能达到60%以上。荧光显微镜和流式细胞仪测定显示,在氮磷比为1∶1、2∶1、5:1、10:1时,转染效率分别为57.8%、64.0%、80.2%、38.3%。在200和500μmol/L枸橼酸铁铵的条件下,MRI检查发现T2加权像(T2WI)的信号均降低,后者更为明显。结论聚乙二醇化树状大分子包裹的纳米金颗粒能够成功地转染目的基因,MRI检查能有效地在体外检测FTH1转染之后的NK92细胞,为MRI活体示踪自然杀伤细胞过继免疫肿瘤治疗奠定了基础。
Objective To transfect the ferritin heavy chain 1(FTH1)into natural killer cell 92(NK92 cells)by a non-viral vector,polyethylene glycol(mPEG)-modified dendrimer-entrapped gold nanoparticles,and to perform magnetic resonance imaging(MRI)in vitro.Methods The fifth generation polyamide amine dendrimer(G5.PAMAM)was coated with mPEG and then entrapped with gold nanoparticles as a non-viral vector,which compressed FTH1 gene transfected into NK92 cells.The synthesized carrier material was characterized by ~1H nuclear magnetic resonance spectroscopy(~1H NMR),ultraviolet spectrophotometer and transmission electron microscopy(TEM).The optimum nitrogen/phosphorus(N/P)ratio between the carrier material and the FTH1 gene was determined by agarose gel electrophoresis.The toxicity of the carrier material was determined by cell counting kit(CCK)-8 kit.The hydrodynamic particle size in different N/P ratios was measured to verify whether the complex of vector/FTH1 could successfully pass through cytomembranes.The transfection efficiency was tested by fluorescence microscope and flow cytometer;and finally MRI was performed in vitro with 3.0 TMRI.Results{(Au~0)_(25)-G5.NH_2-mPEG_(17)}dendrimer-entrapped gold nanoparticles(AuDENPs)with good biocompatibility was synthesized successfully.The cytotoxicity of the material was small,and cell survival rate was up to 60% at the concentration of 3 000 nmol/L.Fluorescence microscopy and flow cytometry showed that the transfection efficiency was 57.8%,64.0%,80.2% and 38.3% at N/P ratio of 1∶1,2∶1,5∶1 and 10∶1,respectively.In the condition of 200 and 500μmol/L ferric ammonium citrate,MRI examination found that T2 weighted imaging(T2WI)signal was significantly reduced,especially in the 500μmol/L ferric ammonium citrate.Conclusion {(Au~0)_(25)-G5.NH_2-mPEG_(17)}DENPs can successfully transfect target genes.MRI can effectively detect NK92 cells after FTH1 transfection invitro,which provides further clinical application of MRI visualization to evaluate the clinical efficacy of NK cell adoptive immunotherapy.
Objective To transfect the ferritin heavy chain 1(FTH1)into natural killer cell 92(NK92 cells)by a non-viral vector,polyethylene glycol(mPEG)-modified dendrimer-entrapped gold nanoparticles,and to perform magnetic resonance imaging(MRI)in vitro.Methods The fifth generation polyamide amine dendrimer(G5.PAMAM)was coated with mPEG and then entrapped with gold nanoparticles as a non-viral vector,which compressed FTH1 gene transfected into NK92 cells.The synthesized carrier material was characterized by ~1H nuclear magnetic resonance spectroscopy(~1H NMR),ultraviolet spectrophotometer and transmission electron microscopy(TEM).The optimum nitrogen/phosphorus(N/P)ratio between the carrier material and the FTH1 gene was determined by agarose gel electrophoresis.The toxicity of the carrier material was determined by cell counting kit(CCK)-8 kit.The hydrodynamic particle size in different N/P ratios was measured to verify whether the complex of vector/FTH1 could successfully pass through cytomembranes.The transfection efficiency was tested by fluorescence microscope and flow cytometer;and finally MRI was performed in vitro with 3.0 TMRI.Results{(Au~0)_(25)-G5.NH_2-mPEG_(17)}dendrimer-entrapped gold nanoparticles(AuDENPs)with good biocompatibility was synthesized successfully.The cytotoxicity of the material was small,and cell survival rate was up to 60% at the concentration of 3 000 nmol/L.Fluorescence microscopy and flow cytometry showed that the transfection efficiency was 57.8%,64.0%,80.2% and 38.3% at N/P ratio of 1∶1,2∶1,5∶1 and 10∶1,respectively.In the condition of 200 and 500μmol/L ferric ammonium citrate,MRI examination found that T2 weighted imaging(T2WI)signal was significantly reduced,especially in the 500μmol/L ferric ammonium citrate.Conclusion {(Au~0)_(25)-G5.NH_2-mPEG_(17)}DENPs can successfully transfect target genes.MRI can effectively detect NK92 cells after FTH1 transfection invitro,which provides further clinical application of MRI visualization to evaluate the clinical efficacy of NK cell adoptive immunotherapy.
引文
[1]CHO I K,MORAN S P,PAUDYAL R,et al.Longitudinal monitoring of stem cell grafts in vivo using magnetic resonance imaging with inducible maga as a genetic reporter[J].Theranostics,2014,4(10):972-989.
[2]KIM H S,WOO J,CHOI Y,et al.Noninvasive MRI and multilineage differentiation capability of ferritin-transduced human mesenchymal stem cells[J].NMR Biomed,2015,28(2):168-179.
[3]PEREIRA S M,HERRMANN A,MOSS D,et al.Evaluating the effectiveness of transferrin receptor-1(TfR1)as a magnetic resonance reporter gene[J].Contrast Media Mol Imaging,2016,11(3):236-244.
[4]NAUMOVA A V,REINECKE H,YARNYKH V,et al.Ferritin overexpression for noninvasive magnetic resonance imaging-based tracking of stem cells transplanted into the heart[J].Mol Imaging,2010,9(4):201-210.
[5]KAY M A,GLORIOSO J C,NALDINI L.Viral vectors for gene therapy:the art of turning infectious agents into vehicles of therapeutics[J].Nat Med,2001,7(1):33-40.
[6]THOMAS C E,EHRHARDT A,KAY M A.Progress and problems with the use of viral vectors for gene therapy[J].Nat Rev Genet,2003,4(5):346-358.
[7]WU J,YAMANOUCHI D,CHU C C.Biodegradable arginine-based poly(ether ester amide)s as a non-viral DNA delivery vector and their structure-function study[J].J Mater Chem,2012,22(36):18983-18991.
[8]PALEOS C M,TZIVELEKA L A,SIDERATOU Z,et al.Gene delivery using functional dendritic polymers[J].Expert Opin Drug Deliv,2009,6(1):27-38.
[9]MCLAUGHLIN R,HYLTON N.MRI in breast cancer therapy monitoring[J].NMR Biomed,2011,24(6):712-720.
[10]HE X,CAI J,LIU B,et al.Cellular magnetic resonance imaging contrast generated by the ferritin heavy chain genetic reporter under the control of a Tet-On switch[J/OL].Stem Cell Res Ther,2015,6:207[2015-10-31].https://stemcellres.biomedcentral.com/articles/10.1186/s13287-015-0205-z.
[11]MORENO-NAVARRETE J M,BLASCO G,XIFRA G,et al.Obesity is associated with gene expression and imaging markers of iron accumulation in skeletal muscle[J].J Clin Endocrinol Metab,2016,101(3):1282-1289.
[12]WANG W,XIONG W,WAN J,et al.The decrease of PAMAM dendrimer-induced cytotoxicity by PEGylation via attenuation of oxidative stress[J].Nanotechnology,2009,20(10):105103.
[13]LEE M,KIM S W.Polyethylene glycol-conjugated copolymers for plasmid DNA delivery[J].Pharm Res,2005,22(1):1-10.
[14]TANG Y,LI Y B,WANG B,et al.Efficient in vitro siRNA delivery and intramuscular gene silencing using PEGmodified PAMAM dendrimers[J].Mol Pharm,2012,9(6):1812-1821.
[15]XU L,SHEN W,WANG B,et al.Efficient siRNA delivery using PEG-conjugated PAMAM dendrimers targeting vascular endothelial growth factor in a CoCl2-induced neovascularization model in retinal endothelial cells[J].Curr Drug Deliv,2016,13(4):590-599.
[16]DUNCAN B,KIM C,ROTELLO V M.Gold nanoparticle platforms as drug and biomacromolecule delivery systems[J].J Control Release,2010,148(1):122-127.
[17]LI D,HE Q,LI J.Smart core/shell nanocomposites:intelligent polymers modified gold nanoparticles[J].Adv Colloid Interface Sci,2009,149(1-2):28-38.
[2]KIM H S,WOO J,CHOI Y,et al.Noninvasive MRI and multilineage differentiation capability of ferritin-transduced human mesenchymal stem cells[J].NMR Biomed,2015,28(2):168-179.
[3]PEREIRA S M,HERRMANN A,MOSS D,et al.Evaluating the effectiveness of transferrin receptor-1(TfR1)as a magnetic resonance reporter gene[J].Contrast Media Mol Imaging,2016,11(3):236-244.
[4]NAUMOVA A V,REINECKE H,YARNYKH V,et al.Ferritin overexpression for noninvasive magnetic resonance imaging-based tracking of stem cells transplanted into the heart[J].Mol Imaging,2010,9(4):201-210.
[5]KAY M A,GLORIOSO J C,NALDINI L.Viral vectors for gene therapy:the art of turning infectious agents into vehicles of therapeutics[J].Nat Med,2001,7(1):33-40.
[6]THOMAS C E,EHRHARDT A,KAY M A.Progress and problems with the use of viral vectors for gene therapy[J].Nat Rev Genet,2003,4(5):346-358.
[7]WU J,YAMANOUCHI D,CHU C C.Biodegradable arginine-based poly(ether ester amide)s as a non-viral DNA delivery vector and their structure-function study[J].J Mater Chem,2012,22(36):18983-18991.
[8]PALEOS C M,TZIVELEKA L A,SIDERATOU Z,et al.Gene delivery using functional dendritic polymers[J].Expert Opin Drug Deliv,2009,6(1):27-38.
[9]MCLAUGHLIN R,HYLTON N.MRI in breast cancer therapy monitoring[J].NMR Biomed,2011,24(6):712-720.
[10]HE X,CAI J,LIU B,et al.Cellular magnetic resonance imaging contrast generated by the ferritin heavy chain genetic reporter under the control of a Tet-On switch[J/OL].Stem Cell Res Ther,2015,6:207[2015-10-31].https://stemcellres.biomedcentral.com/articles/10.1186/s13287-015-0205-z.
[11]MORENO-NAVARRETE J M,BLASCO G,XIFRA G,et al.Obesity is associated with gene expression and imaging markers of iron accumulation in skeletal muscle[J].J Clin Endocrinol Metab,2016,101(3):1282-1289.
[12]WANG W,XIONG W,WAN J,et al.The decrease of PAMAM dendrimer-induced cytotoxicity by PEGylation via attenuation of oxidative stress[J].Nanotechnology,2009,20(10):105103.
[13]LEE M,KIM S W.Polyethylene glycol-conjugated copolymers for plasmid DNA delivery[J].Pharm Res,2005,22(1):1-10.
[14]TANG Y,LI Y B,WANG B,et al.Efficient in vitro siRNA delivery and intramuscular gene silencing using PEGmodified PAMAM dendrimers[J].Mol Pharm,2012,9(6):1812-1821.
[15]XU L,SHEN W,WANG B,et al.Efficient siRNA delivery using PEG-conjugated PAMAM dendrimers targeting vascular endothelial growth factor in a CoCl2-induced neovascularization model in retinal endothelial cells[J].Curr Drug Deliv,2016,13(4):590-599.
[16]DUNCAN B,KIM C,ROTELLO V M.Gold nanoparticle platforms as drug and biomacromolecule delivery systems[J].J Control Release,2010,148(1):122-127.
[17]LI D,HE Q,LI J.Smart core/shell nanocomposites:intelligent polymers modified gold nanoparticles[J].Adv Colloid Interface Sci,2009,149(1-2):28-38.