壳聚糖/纳米颗粒复合物中pcDNA3.1(+)定位的制备和表征

IF 1.6 4区 生物学 Q4 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Iranian Journal of Biotechnology Pub Date : 2022-07-01 DOI:10.30498/ijb.2022.297534.3110
Tohid Piri-Gharaghie, Abbas Doosti, Seyed Abbas Mirzaei
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引用次数: 8

摘要

背景:壳聚糖纳米颗粒(CSNP)正成为将核酸输送到组织中进行基因转移(基因治疗)的一种流行的替代方法。这些可生物降解的纳米载体的大小和形态是可调节的,它们的正电荷允许它们与带负电荷的核酸强烈相互作用。目的:制备和表征pcDNA3.1(+)质粒(pDNA)和CSNP复合物,并确定质粒在这些载体中的位置。材料与方法:采用SEM、XRD、DLS、TGA、FTIR等方法对合成后的pDNA/CSNP配合物进行表征。通过荧光插入染料oliggreen标记游离质粒,研究了CSNP与pDNA形成复合物的能力。评估pDNA/CSNP在壳聚糖酶存在下的稳定性。采用表面增强拉曼光谱(SERS)进行pDNA定位,并采用实时荧光定量PCR评估BALB/c小鼠的吸收率。结果:通过光谱学测定确定质粒复合物形成的最佳pDNA/CSNP比为1:2 (w.w.)。在这些最佳络合物形成比、光谱和凝胶消化实验中,SERS表明部分pDNA存在于络合物的外表面。real-time PCR检测小鼠大腿组织质粒吸收结果显示,pDNA/CSNP剂量为1:2 (w.w.)时,基因吸收率显著高于其他组(P< 0.001)。结论:本研究的发现揭示了pDNA完全适合聚合物纳米结构的递送系统,允许配方调整选择性分布。这种理解将有助于未来对该系统在体外和体内功能的研究。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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Fabrication and Characterization of pcDNA3.1(+) Location within Chitosan/Nanoparticles Complexes for Enhanced Gene Delivery.

Background: Chitosan nanoparticles (CSNP) are becoming a popular alternative for delivering nucleic acids to tissues for gene transfer (gene therapy). The size and morphology of these biodegradable nano-carriers are adjustable, and their positive charge allows them to interact strongly with negatively charged nucleic acids.

Objective: This study aimed to fabricate and characterize pcDNA3.1 (+) plasmid (pDNA) and CSNP complexes and determine the plasmid location in these vehicles.

Materials and methods: The characteristics of the pDNA/CSNP complex after production were investigated by SEM, XRD, DLS, TGA, and FTIR. The capacity of CSNP to form complexes with pDNA was investigated by labeling free plasmids with the fluorescent intercalating dye OliGreen. The stability of pDNA/CSNP in the presence of chitosanase was evaluated. Surface-Enhanced Raman Spectroscopy (SERS) for pDNA localization was performed, and absorption rate in BALB/c mice was assessed by real-time PCR.

Results: The optimum pDNA/CSNP ratio for plasmid complex formation was established to be 1:2 (w.w) by measuring spectroscopy. At these optimum complex formation ratios, spectroscopy, and gel digest experiments, SERS indicated that a part of the pDNA was present on the complex outer surface. The findings of plasmid absorption in mouse thigh tissue by real-time PCR revealed that the rate of gene uptake was significantly greater at a dose of 1:2 (w.w) of pDNA/CSNP than in other groups (P< 0.001).

Conclusions: The findings of this study reveal exactly pDNA fits into polymer nanostructured delivery systems, allowing the formulation to be adjusted for selective distribution. This understanding will aid future research into the system's functioning in vitro and in vivo.

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来源期刊
Iranian Journal of Biotechnology
Iranian Journal of Biotechnology BIOTECHNOLOGY & APPLIED MICROBIOLOGY-
CiteScore
2.60
自引率
7.70%
发文量
20
期刊介绍: Iranian Journal of Biotechnology (IJB) is published quarterly by the National Institute of Genetic Engineering and Biotechnology. IJB publishes original scientific research papers in the broad area of Biotechnology such as, Agriculture, Animal and Marine Sciences, Basic Sciences, Bioinformatics, Biosafety and Bioethics, Environment, Industry and Mining and Medical Sciences.
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