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Chloroplast 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-SWNCT have a statistically significant higher percentage of DNA colocalization events with algal chloroplasts (22.28% ± 6.42, 1 hr) over 1-3 hours than DNA-PEI10k-SWNCT (7.23% ± 0.68, 1 hr) (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/cell) and DNA-PEI10k-SWCNT (300 fg/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/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. 

Photosynthetic organisms are sources of sustainable foods, renewable biofuels, novel biopharmaceuticals, and next-generation biomaterials essential for modern society. Efforts to improve the yield, variety, and sustainability of products dependent on chloroplasts are limited by the need for biotechnological approaches for high-throughput chloroplast transformation, monitoring chloroplast function, and engineering photosynthesis across diverse plant species. The use of nanotechnology has emerged as a novel approach to overcome some of these limitations. Nanotechnology is enabling advances in the targeted delivery of chemicals and genetic elements to chloroplasts, nanosensors for chloroplast biomolecules, and nanotherapeutics for enhancing chloroplast performance. Nanotechnology-mediated delivery of DNA to the chloroplast has the potential to revolutionize chloroplast synthetic biology by allowing transgenes, or even synthesized DNA libraries, to be delivered to a variety of photosynthetic species. Crop yield improvements could be enabled by nanomaterials that enhance photosynthesis, increase tolerance to stresses, and act as nanosensors for biomolecules associated with chloroplast function. Engineering isolated chloroplasts through nanotechnology and synthetic biology approaches are leading to a new generation of plant-based biomaterials able to self-repair using abundant CO 2 and water sources and are powered by renewable sunlight energy. Current knowledge gaps of nanotechnology-enabled approaches for chloroplast biotechnology include precise mechanisms for entry into plant cells and organelles, limited understanding about nanoparticle-based chloroplast transformations, and the translation of lab-based nanotechnology tools to the agricultural field with crop plants. Future research in chloroplast biotechnology mediated by the merging of synthetic biology and nanotechnology approaches can yield tools for precise control and monitoring of chloroplast function in vivo and ex vivo across diverse plant species, allowing increased plant productivity and turning plants into widely available sustainable technologies.