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γ-Ray-Radiation-Scissioned Chitosan as a gene Carrier and Its Improved in vitro gene transfection Performance

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Chinese Journal of Chemical Physics 2017年第2期30卷 231-238,I0002页

作者：林福星曾琨杨文秀汪谟贞荣洁琳谢娟赵宇葛学武中国科学院软物质化学重点实验室中国科学技术大学高分子科学与工程系合肥230026 安徽医科大学第一附属医院整形外科合肥230031

Chitosan （CS） is expected to be an ideal gene carrier for its high biosafety. In this work, CS with low molecular weight were prepared through the γ-ray radiation on the acetic acid solution of CS. The CS chains were scissioned under the γ-ray radiation, and the molecu- lar weight （MW） of CS decreased with the absorbed dose. When the absorbed dose was above 30 kGy, the molecular weight of CS decreased about an order of magnitude. The γ-ray-radiation-scissioned CS can effectively bind with plasmid （pEGFP） through complex coacervation method, forming pEGFP/γ-ray-radiation-scissioned CS complex particles with a size of 200-300 nm. The complex particles have good stability and little cytotoxicity. The in vitro gene transfection efficiencies of the pEGFP/γ-ray-radiation-scissioned CS complex particles were investigated by fluorescence microscope and flow cytometry. The results showed that the gene vectors using γ-ray-radiation-scissioned CS as the carrier will possess better gene transfection efficiency than those using natural high-MW CS as the carrier. The higher the absorbed dose, the smaller the MW of CS and the better transfection efficiency of the corresponding gene vector. This work provides a green and simple method on the preparation of CS-based gene vectors with high efficiency and biosafety.

关键词： Chitosan Biocompatibility Radiation scission gene transfection

Pillar[5]arene-modified peptide-guanidiniocarbonylpyrrol amphiphiles with gene transfection properties

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Chinese Chemical Letters 2023年第5期34卷 246-249页

作者： Kaiya Wang Minzan Zuo Tao Zhang Huilan Yue Xiao-Yu Hu College of Materials Science and Technology Nanjing University of Aeronautics and AstronauticsNanjing 211106China Qinghai Provincial Key Laboratory of Tibetan Medicine Research and Key Laboratory of Tibetan Medicine Research Northwest Institute of Plateau BiologyChinese Academy of SciencesXining 810008China

Pillar[5]arene-modified amphiphilic peptides with varying numbers of guanidiniocarbonylpyrrol(GCP)moieties have been successfully synthesized,which can self-assemble to multivalent cationic superstructures in aqueous *** assembled peptides can condense DNA into various compact multimolecular aggregates to achieve successful intracellular DNA delivery and demonstrate great potential for gene *** efficiencies of the self-assembled superstructures have been evaluated in vitro with He La and HEK 293T *** demonstrate that GCP moiety could enhance the cell transfection ability,owing to its excellent binding towards *** was also found that subtle structure difference in peptides 2 and 3 could result in distinct transfection efficacy,which makes it possible to gain an in-depth understanding of their structure-activity *** work presents a good example of rational structural design in achieving effective gene transfection vectors.

关键词： Pillararene Guanidinocarbonylpyrrole gene transfection Amphiphiles Structure-activity relationship

Efficient gene transfection of suspension cells by highly branched poly(β-amino ester)

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Chinese Chemical Letters 2023年第7期34卷 176-181页

作者： Delu Che Chenfei Wang Zhili Li Kaixuan Wang Shuaiwei Sun Xinyue Zhang Yi Li Zhengju Chen Lei Guo Yajing Hou Dezhong Zhou Songmei Geng Department of Dermatology the Second Hospital Affiliated to Xi'an Jiaotong UniversityXi'an 710061China School of Chemical Engineering and Technology Xi'an Jiaotong UniversityXi'an 710049China Infectious Disease Department the Second Xiangya Hospital of Central South UniversityChangsha 410011China Pooling Medical Research Institutes Hangzhou 310053China Department of Pharmacy Shaanxi Provincial People's HospitalXi'an 710068China

Suspension cells play a crucial role in many biological processes. However, compared to adherent cells, it is particularly challenging to introduce exogenous genes into suspension cells to regulate their biological functions with non-viral gene vectors, mainly due to the low cellular uptake and endosomal escape of polyplexes. Herein, to improve the interactions of polyplexes with cellular membranes, we design and synthesize highly branched poly(β-amino ester)(HPAE) via an “A2 + B4 + C2” Michael addition *** show that branching significantly increases DNA condensation of HPAE, cellular uptake and endosomal escape of HPAE/DNA polyplexes. In mast cells(MCs), HPAE exhibits up to 80-fold higher gene transfection efficiency compared to the corresponding linear poly(β-amino ester)(LPAE) and the leading commercial gene transfection reagents PEI25k, jetPEI, and Lipofectamine 3000, without causing obvious cytotoxicity. Our study establishes a reliable non-viral platform for efficient gene transfection of suspension cells.

关键词： Non-viral vector Highly branched poly(β-amino ester) gene transfection High transfection efficiency Suspension cells Mast cells

Highly branched poly(β-amino ester)s with narrow molecular weight distribution: Fractionation and gene transfection activity

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Chinese Chemical Letters 2023年第3期34卷 383-387页

作者： Chenfei Wang Litao Sun Qiuxia Li Zhili Li Chengyuan Xu Xinyue Zhang Jianjun Shi Hao Zhou Wenxin Wang Dezhong Zhou School of Chemical Engineering and Technology Xi’an Jiaotong UniversityXi’an 710049China Key Laboratory of Water Pollution Treatment&Resource Reuse Hainan Normal UniversityHaikou 571158China State Key Laboratory of Medicinal Chemical Biology Tianjin Key Laboratory of Protein Scienceand College of Life SciencesNankai UniversityTianjin 300071China Department of Dermatology The Second Hospital Affiliated to Xi’an Jiaotong UniversityXi’an 710061China Charles Institute of Dermatology School of MedicineUniversity College DublinDublin 4Ireland

Highly branched poly(β-amino ester)s(HPAEs) have shown their great promise in gene delivery. However, their broad molecular weight distribution(MWD) poses an additional challenge to the mechanistic understanding of the influence of molecular weight(MW) on their gene transfection activity. Using a stepwise precipitation strategy, HPAEs were fractionated. It is shown that MW has a significant effect on the transfection activity and cytotoxicity of HPAEs. The intermediate MW mediates higher transfection efficiency while maintaining high cell viability. Mechanistic studies show that the intermediate MW confers stronger DNA binding affinity to HPAEs, leading to the formulation of polyplexes with a relatively smaller size and more positive zeta potential. This study not only suggests a simple strategy to fractionate HPAEs with narrow MWD but also provides new insights into understanding the structure-property relationship, which would facilitate the clinical translation of HPAEs in gene therapy.

关键词： gene transfection Non-viral vector Highly branched poly(β-amino ester)s Fractionation transfection activity

Ultrasound-targeted cationic microbubble-mediated gene transfection and inhibition of retinal neovascularization

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International Journal of Ophthalmology(English edition) 2022年第6期15卷 876-885页

作者： Ming-Xing Wu Yu Zhou Xi-Yuan Zhou Yan Xu Department of Ophthalmology Second Affiliated Hospital of Chongqing Medical UniversityChongqing 400010China

AIM:To investigate whether ultrasound-targeted cationic microbubbles(CMBs)destruction could deliver endostatingreen fluorescent protein(GFP)plasmids efficiently to the human retinal endothelial cells(HRECs)and inhibit retinal neovascularization in ***:CMBs were prepared and the presentation of GFP reporter was confirmed by flow cytometry and laser confocal *** assessing HRECs migration and vascular formation were per formed to evaluate gene therapy’s efficiency in vitro.A mouse model of oxygen-induced retinopathy was employed and the expression of Bcl-xl,Bcl-2,vascular endothelial growth factor(VEGF)and endostatin in the retina of mice were determined by Western blotting and quantitative polymerase chain reaction(q PCR).The expression of endostatin-GFP in the retina was examined by laser confocal microscopy at 5,14,and 28 d after ***:The gene expression of endostatin was the highest in the group of the ***,the inhibition and antiangiogenesis effect of the migration and development of HRECs were improved following treatment with CMBs compared with the other groups in *** vivo,retinal neovascularization was significantly inhibited and the fluorescence intensity of endostatin-GFP in the mouse retina was importantly higher in the group of CMBs than that in other ***:The research illustrates ultrasoundtargeted CMBs destruction possessed distinct effect on the inhibition of the vascular formation and the development of retinal neovascularization both in vitro and in vivo.

关键词： ultrasound cationic microbubbles human retinal vascular endothelial cells gene transfection retinal neovascularization

gene transfection Mediated by Ultrasound and Pluronic P85 in HepG2 Cells

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2007年第6期27卷 700-702页

作者：王芬李开艳陈云超邓远洪恺 Department of Medical Ultrasound Tongji Hospital Tongji Medical College Huazhong University of Science and Technology

In order to assess whether gene transfection could be mediated by ultrasound in association with P85 and find the appropriate parameters of ultrasound irradiation, the effects of ultrasound with or without P85 on gene transfection of HepG2 cells were examined. The HepG2 cells were irra- diated by ultrasound at 1 MHz, 0.4-2.0 W/cm2 and 50% duty cycle with plasmid encoding enhanced green fluorescent protein （EGFP） as a report gene. Forty-eight h later, the expression of EGFP was detected under the fluorescence microscopy. transfection efficacy was quantitatively assessed by flow cytometry, and cell viability was evaluated by trypan blue exclusion. The results showed that the transfection efficacy was increased with the increases in ultrasound output power and the ideal transfection efficacy was achieved in HepG2 cells irradiated by ultrasound at 0.8 W/cm2 for 30 s. The transfection efficacy in ulstrasound＋P85 group was three times higher than in single ultrasound group [（17.63±1.07）% vs （5.57±0.56）%, P〈0.05]. The cell viability was about 81% and 62% in ultrasound group and ultrasound＋P85 group respectively. It was concluded that ultrasound in combination with P85 could mediate the gene transfection of HepG2 cells, ideal transfection efficacy was achieved by ultrasound irradiation at 0.8 W/cm2 for 30 s, and P85 could somewhat increase the damage to cells caused by ultrasound.

关键词： ultrasound P85 gene transfection HepG2

BASIC FIBROBLAST GROWTH FACTOR gene transfection TO ENHANCE THE REPAIR OF AVASCULAR NECROSIS OFTHE FEMORAL HEAD

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Chinese Medical Sciences Journal 2004年第2期19卷 111-115页

作者： CaoYang Shu-huaYang Jing-yuanDu JinLi Wei-huaXu Yu-fangXiong DepartmentofOrthopedics UnionHospitalHuazhongUniversityofScienceandTechnologyWuhan430022 DepartmentofBiochemistry TongjiMedicalCollegeHuazhongUniversityofScienceandTechnologyWuhan430022

Objective To explore a new method for the therapy of avascular necrosis of the femoral head. Method The recombinant plasmid pCD-rbFGF was mixed with collagen and was implanted in the necrotic femoral head. Expression of basic fibroblast growth factor (bFGF) was examined by RT-PCR and immunohistochemical method. Re-pair of the femoral head was observed by histological and histomorphometric analysis. Result Expression of bFGF was detected in the femoral head transfected with bFGF gene, indicating significant increase of angiogenesis 2 weeks after gene transfection and increased new bone formation 8 weeks after gene transfection on histom-orphometric analysis (Ptransfection of bFGF gene enhances bone tissue angiogenesis. Repair in osteonecrosis would be accelerated accordingly.

关键词： basic fibroblast growth factor gene transfection avascular necrosis femoral head

Heat shock protein 70 gene transfection protects rat myocardium cell against anoxia-reoxygeneration injury

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Chinese Medical Journal 2007年第7期120卷 578-583页

作者： LIU Ji-chun HE Ming WAN Li CHENG Xiao-shu Department of Cardiovascular Surgery First Affiliated Hospital of Nanchang University Nanchang 330006 China Department of Pharmacology Medical College of Nanchang University Nanchang 330006 China Department of Cardiovascular Medicine Second Affiliated Hospital of Nanchang University Nanchang 330006 China

Background A number of studies suggest that the expression of heat shock protein 70 （HSP70） induced by heat stress are associated with protection against ischemia-reperfusion injury. But the protective effects may be contaminated by other factors in the same stress. This study was conducted to explore the protective role of HSP70 expression in acute myocardial anoxia/reoxygeneration （A/R） injury with a liposome-mediated gene transfer technique for the introduction of pCDNA HSP70 into the neonatal rat myocardial cells. In addition, heat shock stress cytoprotection was also investigated for comparison. Methods The cultured primary neonatal rat myocardiocytes with an acute myocardial A/R injury model and the HS-treated rat myocardiocyte model were used. Three-day cultured myocardiocytes were randomly divided into four groups （n=8）： control group, A/R group, HS＋A/R group and pCDNA HSP70 ＋A/R group. A liposome-coated HSP70 pCDNA plasmid was transfected into the primary neonatal rat myocardiocytes; HSP70 mRNA and its protein were confirmed by reverse transcriptase polymerase chain reaction （RT-PCR） and Western blotting. The cell viability was assayed by monotetrazolium （MTT） and the lactate dehydrogenase （LDH） and creatine phosphokinase （CPK） activity of cells during incubation and the changes in cells ultrastructure were examined. NF-κB activity in the primary neonatal rat myocardiocytes was measured with flow cytometry. Results Compared with viability in the A/R group （（35.4±6.9）%） the cell viability in the HS＋A/R group （（72.8±11.6）%） and the pCDNA HSP70 ＋ A/R group （（76.3±12.2）%） was improved significantly （P〈0.05）. The activity of LDH and CPK was significantly elevated in the A/R group. However, in the HS＋A/R group and pCDNA HSP70 ＋A/R group, significant decreases in activity were observed. The cell ultrastructure of the A/R group cells was abnormal, whereas nearly normal ultrastructure was observed in HS＋A/R group and pCDNA HSP70＋A/R group. HSP70 mRNA and protein

关键词： gene transfection HSP70 gene NF-κB cardiac myocyte anoxia-reoxygeneration injury

Brain-derived neurotrophic factor gene transfection promotes neuronal repair and neurite regeneration after diffuse axonal injury

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Neural Regeneration Research 2011年第25期6卷 1942-1946页

作者： Yin Yu Xingli Zhao Jiajia Shao Qiang Shen Tao Jiang Wei WU Dong Zhu Yu Tian Yongchuan Gu Second Department of Neurosurgery Bethune Third Hospital China-Japan Friendship Hospital Jilin University Changchun 130031 JilinProvince China Changchun Vocational Institute of Technology Changchun 130031 Jilin Province China Department of Neurosurgery Jilin Provincial People＇s Hospital Changchun 130000 Jilin Province China Department of Neurosurgery Bethune First Hospital Jilin University Changchun 130021 Jilin Province China

This study sought to assess the potential of brain-derived neurotrophic factor （BDNF） to promote neuronal repair and regeneration in rats with diffuse axonal injury, and to examine the accompanying neurobiological changes. BDNF gene transfection reduced the severity of the pathological changes associated with diffuse axonal injury in cortical neurons of the frontal lobe and increased neurofilament protein expression. These findings demonstrate that BDNF can effectively promote neuronal repair and neurite regeneration after diffuse axonal injury.

关键词： diffuse axonal injury brain-derived neurotrophic factor neurite gene transfection neural regeneration