DNA delivery by high aspect ratio nanomaterials to algal chloroplasts
DNA delivery by high aspect ratio nanomaterials to algal chloroplasts
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高纵横比纳米材料将 DNA 递送至藻类叶绿体
DOI：
10.1039/d3en00268c
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Giraldo, Juan Pablo
中科院分区：
文献类型：
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作者：
Newkirk, Gregory M.;Jeon, Su-Ji;Kim, Hye-In;Sivaraj, Supreetha;De Allende, Pedro;Castillo, Christopher;Jinkerson, Robert E.;Giraldo, Juan Pablo
叶绿体是光合作用的场所，已经被生物工程化以生产食物、生物药物和生物材料。目前用于改变叶绿体基因组的方法依赖于低效的DNA递送方法，导致叶绿体转化效率低。对于藻类叶绿体，没有可修改的、可定制的和有效的原位DNA递送底盘。在此，我们研究了聚乙烯亚胺包覆的单壁碳纳米管（PEI-SWCNT）作为DNA到藻类叶绿体的递送载体。我们研究了PEI-SWCNT电荷和PEI聚合物尺寸（25 k对10 k）对绿色藻类莱茵衣藻的野生型和细胞壁敲除突变体菌株的叶绿体摄取的影响。为了评估与PEI-SWCNT结合的DNA的递送，我们使用了叶绿体自发荧光与荧光团标记的单链GT 15 DNA的共聚焦显微镜和共定位分析。我们发现高电荷DNA-PEI 25 k-SWCNT在1-3 h内与藻叶绿体的DNA共定位事件百分比（22.28% ± 6.42，1 h）显著高于DNA-PEI 10 k-SWCNT（7.23% ± 0.68，1 h）（P < 0.01）。我们通过活藻细胞、活性氧（ROS）生成和体内叶绿素测定确定DNA-PEI-SWCNT的生物相容性。通过这些测定，表明暴露于DNA-PEI 25 k-SWCNT（30 fg/细胞）和DNA-PEI 10 k-SWCNT（300 fg/细胞）的藻类在4天内是存活的，并且对氧化应激水平几乎没有影响。在野生型菌株和细胞壁敲除菌株中，DNA包被的PEI-SWCNT在暴露于纳米材料（30-300 fg/细胞）的一小时内瞬时增加ROS水平，随后由于与抗氧化剂谷胱甘肽和脂质膜的反应，ROS下降至正常水平。PEI-SWCNT可以作为生物载体传递生物分子，如DNA，并有可能成为叶绿体生物技术和合成生物学的新工具。
Chloroplasts are sites of photosynthesis that have been bioengineered to produce food, biopharmaceuticals, and biomaterials. Current approaches for altering the chloroplast genome rely on inefficient DNA delivery methods, leading to low chloroplast transformation efficiency rates. For algal chloroplasts, there is no modifiable, customizable, and efficient in situ DNA delivery chassis. Herein, we investigated polyethylenimine-coated single-walled carbon nanotubes (PEI-SWCNT) as delivery vehicles for DNA to algal chloroplasts. We examined the impact of PEI-SWCNT charge and PEI polymer size (25k vs. 10k) on the uptake into chloroplasts of wildtype and cell wall knockout mutant strains of the green algae Chlamydomonas reinhardtii. To assess the delivery of DNA bound to PEI-SWCNT, we used confocal microscopy and colocalization analysis of chloroplast autofluorescence with fluorophore-labeled single-stranded GT15 DNA. We found that highly charged DNA–PEI25k-SWCNT have a statistically significant higher percentage of DNA colocalization events with algal chloroplasts (22.28% ± 6.42, 1 h) over 1–3 hours than DNA–PEI10k-SWCNT (7.23% ± 0.68, 1 h) (P < 0.01). We determined the biocompatibility of DNA–PEI-SWCNT through assays for living algae cells, reactive oxygen species (ROS) generation, and in vivo chlorophyll assays. Through these assays, it was shown that algae exposed to DNA–PEI25k-SWCNT (30 fg per cell) and DNA–PEI10k-SWCNT (300 fg per cell) were viable over 4 days and had little impact on oxidative stress levels. DNA coated PEI-SWCNT transiently increased ROS levels within one hour of exposure to nanomaterials (30–300 fg per cell) both in the wildtype strain and cell wall knockout strain, followed by ROS decline to normal levels due to reaction with antioxidant glutathione and lipid membranes. PEI-SWCNT can act as biological carriers for delivering biomolecules such as DNA and have the potential to become novel tools for chloroplast biotechnology and synthetic biology.