Agriculture is facing new challenges, with global warming modifying the survival chances for crops, and new pests on the horizon. To keep up with these challenges, gene delivery provides tools to increase crop yields. On the other hand, gene delivery also opens the door for molecular farming of pharmaceuticals in plants. However, towards increased food production and scalable molecular farming, there remain technical difficulties and regulatory hurdles to overcome. The industry-standard is transformation of plants via Agrobacterium tumefaciens , but this method is limited to certain plants, requires set up of plant growth facilities and fermentation of bacteria, and introduces lipopolysaccharides contaminants into the system. Therefore, alternate methods are needed. Mechanical inoculation and spray methods have already been discussed in the literature – and here, we compare these methods with a newly introduced petiole injection technique. Because our interest lies in the development of plant viruses as immunotherapies targeting human health as well as gene delivery vectors for agriculture applications, we turned toward tobacco mosaic virus as a model system. We studied the effectiveness of three inoculation techniques: mechanical inoculation, Silwet-77 foliar spray and petiole injections. The foliar spray method was optimized, and we used 0.03% Silwet L-77 to induce infection using either TMV or a lysine-added mutant TMV-Lys. We developed a method using a needle-laden syringe to target and inject the plant virus directly into the vasculature of the plant – we tested injection into the stem and petiole. Stem inoculation resulted in toxicity, but the petiole injection technique was established as a viable strategy. TMV and TMV-Lys were purified from single plants and pooled leaf samples – overall there was little variation between the techniques, as measured by TMV or TMV-Lys yields, highlighting the feasibility of the syringe injection technique to produce virus nanoparticles. There was variation between yields from preparation to preparation with mechanical, spray and syringe inoculation yielding 40–141 mg, 36–56 mg, 18–56 mg TMV per 100 grams of leaves. Similar yields were obtained using TMV-Lys, with 24–38 mg, 17–28, 7–36 mg TMV-Lys per 100 grams of leaves for mechanical, spray and syringe inoculation, respectively. Each method has its advantages: spray inoculation is highly scalable and therefore may find application for farming, the syringe inoculation could provide a clean, aseptic, and controlled approach for molecular farming of pharmaceuticals under good manufacturing protocols (GMP) and would even be applicable for gene delivery to plants in space.
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Integrating plant molecular farming and materials research for next-generation vaccines
Biologics — medications derived from a biological source — are increasingly used
as pharmaceuticals, for example, as vaccines. Biologics are usually produced in bacterial, mammalian or insect cells. Alternatively, plant molecular farming, that is, the manufacture of biologics in plant cells, transgenic plants and algae, offers a cheaper and easily adaptable strategy for the production of biologics, in particular, in low- resource settings. In this Review, we discuss current vaccination challenges, such as cold chain requirements, and highlight how plant molecular farming in combination with advanced materials can be applied to address these challenges. The production of plant viruses and virus- based nanotechnologies in plants enables low- cost and regional fabrication of thermostable vaccines. We also highlight key new vaccine delivery technologies, including microneedle patches and material platforms for intranasal and oral delivery. Finally, we provide an outlook of future possibilities for plant molecular farming of next- generation vaccines and biologics.
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- Award ID(s):
- 2011924
- NSF-PAR ID:
- 10324276
- Date Published:
- Journal Name:
- Nature Reviews Materials
- ISSN:
- 2058-8437
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Background Agriculture faces significant global challenges including climate change and an increasing food demand due to a growing population. Addressing these challenges will require the adoption of transformative innovations into biotechnology practice, such as nanotechnology. Recently, nanomaterials have emerged as unmatched tools for their use as biosensors, or as biomolecule delivery vehicles. Despite their increasingly prolific use, plant-nanomaterial interactions remain poorly characterized, drawing into question the breadth of their utility and their broader environmental compatibility.
Results Herein, we characterize the response of
Arabidopsis thaliana to single walled carbon nanotube (SWNT) exposure with two different surface chemistries commonly used for biosensing and nucleic acid delivery: oligonucleotide adsorbed-pristine SWNTs, and polyethyleneimine-SWNTs loaded with plasmid DNA (PEI-SWNTs), both introduced by leaf infiltration. We observed that pristine SWNTs elicit a mild stress response almost undistinguishable from the infiltration process, indicating that these nanomaterials are well-tolerated by the plant. However, PEI-SWNTs induce a much larger transcriptional reprogramming that involves stress, immunity, and senescence responses. PEI-SWNT-induced transcriptional profile is very similar to that of mutant plants displaying a constitutive immune response or treated with stress-priming agrochemicals. We selected molecular markers from our transcriptomic analysis and identified PEI as the main cause of this adverse reaction. We show that PEI-SWNT response is concentration-dependent and, when persistent over time, leads to cell death. We probed a panel of PEI variant-functionalized SWNTs across two plant species and identified biocompatible SWNT surface functionalizations.Conclusions While SWNTs themselves are well tolerated by plants, SWNTs surface-functionalized with positively charged polymers become toxic and produce cell death. We use molecular markers to identify more biocompatible SWNT formulations. Our results highlight the importance of nanoparticle surface chemistry on their biocompatibility and will facilitate the use of functionalized nanomaterials for agricultural improvement.
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Abstract Cancer vaccines hold promise as an immunotherapeutic modality based on their potential to generate tumor antigen-specific T cell responses and long-lived antitumor responses capable of combating metastatic disease and recurrence. However, cancer vaccines have historically failed to deliver significant therapeutic benefit in the clinic, which we maintain is due in part to drug delivery challenges that have limited vaccine immunogenicity and efficacy. In this review, we examine some of the known and putative failure mechanisms of common first-generation clinical cancer vaccines, and describe how the rational design of materials engineered for vaccine delivery and immunomodulation can address these shortcomings. First, we outline vaccine design principles for augmenting cellular immunity to tumor antigens and describe how well-engineered materials can improve vaccine efficacy, highlighting recent innovations in vaccine delivery technology that are primed for integration into neoantigen vaccine development pipelines. We also discuss the importance of sequencing, timing, and kinetics in mounting effective immune responses to cancer vaccines, and highlight examples of materials that potentiate antitumor immunity through spatiotemporal control of immunomodulation. Furthermore, we describe several engineering strategies for improving outcomes of in situ cancer vaccines, which leverage local, intratumoral delivery to stimulate systemic immunity. Finally, we highlight recent innovations leveraging nanotechnology for increasing the immunogenicity of the tumor microenvironment (TME), which is critical to enhancing tumor infiltration and function of T cells elicited in response to cancer vaccines. These immunoengineering strategies and tools complement ongoing advances in cancer vaccines as they reemerge as an important component of the immunotherapeutic armamentarium.
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null (Ed.)Nanotechnology platforms, such as nanoparticles, liposomes, dendrimers, and micelles have been studied extensively for various drug deliveries, to treat or prevent diseases by modulating physiological or pathological processes. The delivery drug molecules range from traditional small molecules to recently developed biologics, such as proteins, peptides, and nucleic acids. Among them, proteins have shown a series of advantages and potential in various therapeutic applications, such as introducing therapeutic proteins due to genetic defects, or used as nanocarriers for anticancer agents to decelerate tumor growth or control metastasis. This review discusses the existing nanoparticle delivery systems, introducing design strategies, advantages of using each system, and possible limitations. Moreover, we will examine the intracellular delivery of different protein therapeutics, such as antibodies, antigens, and gene editing proteins into the host cells to achieve anticancer effects and cancer vaccines. Finally, we explore the current applications of protein delivery in anticancer treatments.more » « less
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Targeted delivery of nanomaterials with chemical cargoes in plants enabled by a biorecognition motifmore » « less
Abstract Current approaches for nanomaterial delivery in plants are unable to target specific subcellular compartments with high precision, limiting our ability to engineer plant function. We demonstrate a nanoscale platform that targets and delivers nanomaterials with biochemicals to plant photosynthetic organelles (chloroplasts) using a guiding peptide recognition motif. Quantum dot (QD) fluorescence emission in a low background window allows confocal microscopy imaging and quantitative detection by elemental analysis in plant cells and organelles. QD functionalization with β-cyclodextrin molecular baskets enables loading and delivery of diverse chemicals, and nanoparticle coating with a rationally designed and conserved guiding peptide targets their delivery to chloroplasts. Peptide biorecognition provides high delivery efficiency and specificity of QD with chemical cargoes to chloroplasts in plant cells in vivo (74.6 ± 10.8%) and more specific tunable changes of chloroplast redox function than chemicals alone. Targeted delivery of nanomaterials with chemical cargoes guided by biorecognition motifs has a broad range of nanotechnology applications in plant biology and bioengineering, nanoparticle-plant interactions, and nano-enabled agriculture.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1038/s41578-021-00399-5
