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In this work, we introduce a 3D-printable virus-like particle (VLP)-enhanced cross-linked biopolymer system. VLPs displaying surface-available acrylate groups were prepared through aza-Michael addition to serve as resins. The VLP resins were then photopolymerized into a poly(ethylene glycol) diacrylate (PEGDA) network following DLP 3D printing. This approach represents a convergence of disciplines, where the synergistic interaction between virology and additive manufacturing unlocks new frontiers in biotechnology.
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Tobacco mild green mosaic virus (TMGMV)-like nanocarriers were designed for gene delivery to plant cells. High aspect ratio TMGMVs were coated with a polycationic biopolymer, poly(allylamine) hydrochloride (PAH), to generate highly charged nanomaterials (TMGMV-PAH; 56.20 ± 4.7 mV) that efficiently load (1:6 TMGMV:DNA mass ratio) and deliver single-stranded and plasmid DNA to plant cells. The TMGMV-PAH were taken up through energy-independent mechanisms in Arabidopsis protoplasts. TMGMV-PAH delivered a plasmid DNA encoding a green fluorescent protein (GFP) to the protoplast nucleus (70% viability), as evidenced by GFP expression using confocal microscopy and Western blot analysis. TMGMV-PAH were inactivated (iTMGMV-PAH) using UV cross-linking to prevent systemic infection in intact plants. Inactivated iTMGMV-PAH-mediated pDNA delivery and gene expression of GFP in vivo was determined using confocal microscopy and RT-qPCR. Virus-like nanocarrier-mediated gene delivery can act as a facile and biocompatible tool for advancing genetic engineering in plants.
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Arabidopsis , Proteínas Fluorescentes Verdes , Arabidopsis/virología , Arabidopsis/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Técnicas de Transferencia de Gen , Plásmidos/genética , Poliaminas/química , Protoplastos/metabolismo , Nanoestructuras/química , ADN/química , ADN/administración & dosificaciónRESUMEN
Nanocarriers (NCs) that can precisely deliver active agents, nutrients and genetic materials into plants will make crop agriculture more resilient to climate change and sustainable. As a research field, nano-agriculture is still developing, with significant scientific and societal barriers to overcome. In this Review, we argue that lessons can be learned from mammalian nanomedicine. In particular, it may be possible to enhance efficiency and efficacy by improving our understanding of how NC properties affect their interactions with plant surfaces and biomolecules, and their ability to carry and deliver cargo to specific locations. New tools are required to rapidly assess NC-plant interactions and to explore and verify the range of viable targeting approaches in plants. Elucidating these interactions can lead to the creation of computer-generated in silico models (digital twins) to predict the impact of different NC and plant properties, biological responses, and environmental conditions on the efficiency and efficacy of nanotechnology approaches. Finally, we highlight the need for nano-agriculture researchers and social scientists to converge in order to develop sustainable, safe and socially acceptable NCs.
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The foundation of most food production systems underpinning global food security is the careful management of soil resources. Embedded in the concept of soil health is the impact of diverse soil-borne pests and pathogens, and phytoparasitic nematodes represent a particular challenge. Root-knot nematodes and cyst nematodes are severe threats to agriculture, accounting for annual yield losses of US$157 billion. The control of soil-borne phytoparasitic nematodes conventionally relies on the use of chemical nematicides, which can have adverse effects on the environment and human health due to their persistence in soil, plants, and water. Nematode-resistant plants offer a promising alternative, but genetic resistance is species-dependent, limited to a few crops, and breeding and deploying resistant cultivars often takes years. Novel approaches for the control of phytoparasitic nematodes are therefore required, those that specifically target these parasites in the ground whilst minimizing the impact on the environment, agricultural ecosystems, and human health. In addition to the development of next-generation, environmentally safer nematicides, promising biochemical strategies include the combination of RNA interference (RNAi) with nanomaterials that ensure the targeted delivery and controlled release of double-stranded RNA. Genome sequencing has identified more than 75 genes in root knot and cyst nematodes that have been targeted with RNAi so far. But despite encouraging results, the delivery of dsRNA to nematodes in the soil remains inefficient. In this review article, we describe the state-of-the-art RNAi approaches targeting phytoparasitic nematodes and consider the potential benefits of nanotechnology to improve dsRNA delivery.
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Protein cages are promising tools for the controlled delivery of therapeutics and imaging agents when endowed with programmable disassembly strategies. Here, we produced hybrid nanocomposites made of tobacco mosaic virus (TMV) and magnetic iron oxide nanoparticles (IONPs), designed to disrupt the viral protein cages using magnetically induced release of heat. We studied the effects of this magnetic hyperthermia on the programmable viral protein capsid disassembly using (1) elongated nanocomposites of TMV coated heterogeneously with magnetic iron oxide nanoparticles (TMV@IONPs) and (2) spherical nanocomposites of polystyrene (PS) on which we deposited presynthesized IONPs and TMV via layer-by-layer self-assembly (PS@IONPs/TMV). Notably, we found that the extent of the disassembly of the protein cages is contingent upon the specific absorption rate (SAR) of the magnetic nanoparticles, that is, the heating efficiency, and the relative position of the protein cage within the nanocomposite concerning the heating sources. This implies that the spatial arrangement of components within the hybrid nanostructure has a significant impact on the disassembly process. Understanding and optimizing this relationship will contribute to the critical spatiotemporal control for targeted drug and gene delivery using protein cages.
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Ensayo de Materiales , Nanocompuestos , Tamaño de la Partícula , Virus del Mosaico del Tabaco , Virus del Mosaico del Tabaco/química , Nanocompuestos/química , Materiales Biocompatibles/químicaRESUMEN
Ovarian cancer (OvCa) is a leading cause of mortality among gynecological malignancies and usually manifests as intraperitoneal spheroids that generate metastases, ascites, and an immunosuppressive tumor microenvironment. In this study, we explore the immunomodulatory properties of cowpea mosaic virus (CPMV) as an adjuvant immunotherapeutic agent using an in vitro model of OvCa peritoneal spheroids. Previous findings highlighted the potent efficacy of intratumoral CPMV against OvCa in mouse tumor models. Leveraging the precision control over material deposition and cell patterning afforded by digital-light-processing (DLP) based bioprinting, we constructed OvCa-macrophage spheroids to mimic peritoneal spheroids using gelatin methacrylate (GelMA), a collagen-derived photopolymerizable biomaterial to mimic the extracellular matrix. Following CPMV treatment, bioprinted spheroids exhibited inhibited OvCa progression mediated by macrophage activation. Our analysis indicates that CPMV regulates and activates macrophage to both induce OvCa cell killing and restore normal cell-cell junctions. This study deepened our understanding of the mechanism of CPMV intratumoral immunotherapy in the setting of OvCa. This study also highlights the potential of studying immunotherapies using high throughput tissue models via DLP bioprinting.
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Bioimpresión , Comovirus , Neoplasias Ováricas , Esferoides Celulares , Femenino , Neoplasias Ováricas/patología , Neoplasias Ováricas/virología , Esferoides Celulares/patología , Esferoides Celulares/virología , Bioimpresión/métodos , Animales , Humanos , Ratones , Línea Celular Tumoral , Inmunoterapia/métodos , Macrófagos/virologíaRESUMEN
Plants, essential for food, oxygen, and economic stability, are under threat from human activities, biotic threats, and climate change, requiring rapid technological advancements for protection. Biohybrid systems, merging synthetic macromolecules with biological components, have provided improvement to biological systems in the past, namely, in the biomedical arena, motivating an opportunity to enhance plant well-being. Nevertheless, strategies for plant biohybrid systems remain limited. In this study, we present a method using grafting-from ring-opening metathesis polymerization (ROMP) under physiological conditions to integrate norbornene-derived polymers into live plants by spray coating. The approach involves creating biological macroinitiators on leaf surfaces, which enable subsequent polymerization of norbornene-derived monomers. Characterization techniques, including FTIR spectroscopy, SEM EDS imaging, ICP-MS, nanoindentation, and XPS, confirmed the presence and characterized the properties of the polymeric layers on leaves. The demonstrated modifiability and biocompatibility could offer the potential to maintain plant health in various applications, including the development of thermal barriers, biosensors, and crop protection layers.
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Norbornanos , Hojas de la Planta , Norbornanos/química , Hojas de la Planta/química , Polimerizacion , Polímeros/química , PlásticosRESUMEN
The dramatic effectiveness of recent mRNA (mRNA)-based COVID vaccines delivered in lipid nanoparticles has highlighted the promise of mRNA therapeutics in general. In this report, we extend our earlier work on self-amplifying mRNAs delivered in spherical in vitro reconstituted virus-like particles (VLPs), and on drug delivery using cylindrical virus particles. In particular, we carry out separate in vitro assemblies of a self-amplifying mRNA gene in two different virus-like particles: one spherical, formed with the capsid protein of cowpea chlorotic mottle virus (CCMV), and the other cylindrical, formed from the capsid protein of tobacco mosaic virus (TMV). The mRNA gene is rendered self-amplifying by genetically fusing it to the RNA-dependent RNA polymerase (RdRp) of Nodamura virus, and the relative efficacies of cell uptake and downstream protein expression resulting from their CCMV- and TMV-packaged forms are compared directly. This comparison is carried out by their transfections into cells in culture: expressions of two self-amplifying genes, enhanced yellow fluorescent protein (EYFP) and Renilla luciferase (Luc), packaged alternately in CCMV and TMV VLPs, are quantified by fluorescence and chemiluminescence levels, respectively, and relative numbers of the delivered mRNAs are measured by quantitative real-time PCR. The cellular uptake of both forms of these VLPs is further confirmed by confocal microscopy of transfected cells. Finally, VLP-mediated delivery of the self-amplifying-mRNA in mice following footpad injection is shown by in vivo fluorescence imaging to result in robust expression of EYFP in the draining lymph nodes, suggesting the potential of these plant virus-like particles as a promising mRNA gene and vaccine delivery modality. These results establish that both CCMV and TMV VLPs can deliver their in vitro packaged mRNA genes to immune cells and that their self-amplifying forms significantly enhance in situ expression. Choice of one VLP (CCMV or TMV) over the other will depend on which geometry of nucleocapsid is self-assembled more efficiently for a given length and sequence of RNA, and suggests that these plant VLP gene delivery systems will prove useful in a wide variety of medical applications, both preventive and therapeutic.
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Proteínas de la Cápside , ARN Mensajero , Virus del Mosaico del Tabaco , Animales , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratones , Virus del Mosaico del Tabaco/genética , Proteínas de la Cápside/genética , Bromovirus/genética , Nanopartículas/química , Humanos , Femenino , Vacunas contra la COVID-19/administración & dosificación , Virión/genética , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/genética , Vacunas de Partículas Similares a Virus/administración & dosificación , LiposomasRESUMEN
Chemical pesticide delivery is a fundamental aspect of agriculture. However, the extensive use of pesticides severely endangers the ecosystem because they accumulate on crops, in soil, as well as in drinking and groundwater. New frontiers in nano-engineering have opened the door for precision agriculture. We introduced Tobacco mild green mosaic virus (TMGMV) as a viable delivery platform with a high aspect ratio and favorable soil mobility. In this work, we assess the use of TMGMV as a chemical nanocarrier for agriculturally relevant cargo. While plant viruses are usually portrayed as rigid/solid structures, these are "dynamic materials," and they "breathe" in solution in response to careful adjustment of pH or bathing media [e.g., addition of solvent such as dimethyl sulfoxide (DMSO)]. Through this process, coat proteins (CPs) partially dissociate leading to swelling of the nucleoprotein complexes-allowing for the infusion of active ingredients (AI), such as pesticides [e.g., fluopyram (FLP), clothianidin (CTD), rifampicin (RIF), and ivermectin (IVM)] into the macromolecular structure. We developed a "breathing" method that facilitates inter-coat protein cargo loading, resulting in up to ~ 1000 AIs per virion. This is of significance since in the agricultural setting, there is a need to develop nanoparticle delivery strategies where the AI is not chemically altered, consequently avoiding the need for regulatory and registration processes of new compounds. This work highlights the potential of TMGMV as a pesticide nanocarrier in precision farming applications; the developed methods likely would be applicable to other protein-based nanoparticle systems.
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Plaguicidas , Virus del Mosaico del Tabaco , Tobamovirus , Ecosistema , Plaguicidas/metabolismo , Suelo , ViriónRESUMEN
The key challenge in cancer treatment is prevention of metastatic disease which is therapeutically resistant and carries poor prognoses necessitating efficacious prophylactic approaches that prevent metastasis and recurrence. It is previously demonstrated that cowpea mosaic virus (CPMV) induces durable antitumor responses when used in situ, i.e., intratumoral injection. As a new direction, it is showed that CPMV demonstrates widespread effectiveness as an immunoprophylactic agent - potent efficacy is demonstrated in four metastatic models of colon, ovarian, melanoma, and breast cancer. Systemic administration of CPMV stimulates the innate immune system, enabling attack of cancer cells; processing of the cancer cells and associated antigens leads to systemic, durable, and adaptive antitumor immunity. Overall, CPMV demonstrated broad efficacy as an immunoprophylactic agent in the rejection of metastatic cancer.
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Comovirus , Animales , Ratones , Femenino , Metástasis de la Neoplasia/prevención & control , Humanos , Línea Celular Tumoral , Modelos Animales de EnfermedadRESUMEN
Due to the increasing incidence of cancer, there is a need to develop new platforms that can combat this disease. Cancer immunotherapy is a platform that takes advantage of the immune system to recognize and eradicate tumors and metastases. Our lab has identified a plant virus nanoparticle, cowpea mosaic virus (CPMV) as a promising approach for cancer immunotherapy. When administered intratumorally, CPMV relieves the immune system of tumor-induced immunosuppression and reprograms the tumor microenvironment into an activated state to launch systemic antitumor immunity. The efficacy of CPMV has been tested in many tumor models and in canine cancer patients with promising results: tumor shrinkage, systemic efficacy (abscopal effect), and immune memory to prevent recurrence. To translate this drug candidate from the bench to the clinic, studies that investigate the safety, pharmacology, and toxicity are needed. In this work, we describe the efficacy of CPMV against a metastatic ovarian tumor model and investigate the biodistribution of CPMV after single or repeated intraperitoneal administration in tumor-bearing and healthy mice. CPMV shows good retention in the tumor nodules and broad bioavailability with no apparent organ toxicity based on histopathology. Data indicate persistence of the viral RNA, which remains detectable 2 weeks post final administration, a phenomenon also observed with some mammalian viral infections. Lastly, while protein was not detected in stool or urine, RNA was shed through excretion from mice; however, there was no evidence that RNA was infectious to plants. Taken together, the data indicate that systemic administration results in broad bioavailability with no apparent toxicity. While RNA is shed from the subjects, data suggest agronomical safety. This data is consistent with prior reports and provides support for translational efforts.
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We have developed nanoparticle formulations targeting M2 macrophages for cancer immunotherapy by conjugating high-affinity binding peptides to cowpea mosaic virus as an immunostimulatory adjuvant. We confirmed the targeting of and uptake by M2 macrophages in vitro and the therapeutic efficacy of the nanoparticles against murine melanoma in vivo.
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PEGylation has been the 'gold standard' in bioconjugation due to its ability to improve the pharmacokinetics and pharmacodynamics of native proteins. However, growing clinical evidence of hypersensitivity reactions to PEG due to pre-existing anti-PEG antibodies in healthy humans have raised concerns. Advancements in controlled polymerization techniques and conjugation chemistries have paved the way for the development of protein-polymer conjugates that can circumvent these adverse reactions while retaining the benefits of such modifications. Herein, we show the development of polynorbornene based bioconjugates of therapeutically relevant urate oxidase (UO) enzymes used in the treatment of gout synthesized by grafting-from ring-opening metathesis polymerization (ROMP). Notably, these conjugates exhibit comparable levels of bioactivity to PEGylated UO and demonstrate increased stability across varying temperatures and pH conditions. Immune recognition of conjugates by anti-UO antibodies reveal low protein immunogenicity following the conjugation process. Additionally, UO conjugates employing zwitterionic polynorbornene successfully avoid recognition by anti-PEG antibodies, further highlighting a potential replacement for PEG.
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Plásticos , Urato Oxidasa , Humanos , Polimerizacion , ProteínasRESUMEN
Immune checkpoint therapy (ICT) for cancer can yield dramatic clinical responses; however, these may only be observed in a minority of patients. These responses can be further limited by subsequent disease recurrence and resistance. Combination immunotherapy strategies are being developed to overcome these limitations. We have previously reported enhanced efficacy of combined intratumoral cowpea mosaic virus immunotherapy (CPMV IIT) and ICT approaches. Lymphocyte-activation gene-3 (LAG-3) is a next-generation inhibitory immune checkpoint with broad expression across multiple immune cell subsets. Its expression increases on activated T cells and contributes to T cell exhaustion. We observed heightened efficacy of a combined CPMV IIT and anti-LAG-3 treatment in a mouse model of melanoma. Further, LAG-3 expression was found to be increased within the TME following intratumoral CPMV administration. The integration of CPMV IIT with LAG-3 inhibition holds significant potential to improve treatment outcomes by concurrently inducing a comprehensive anti-tumor immune response, enhancing local immune activation, and mitigating T cell exhaustion.
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Comovirus , Melanoma , Humanos , Animales , Ratones , Terapia Combinada , Inmunoterapia , Modelos Animales de Enfermedad , Melanoma/terapiaRESUMEN
Implantable polymeric hydrogels loaded with immunostimulatory cowpea mosaic virus (CPMV) were fabricated using digital light processing (DLP) printing technology. The CPMV-laden hydrogels were surgically implanted into the peritoneal cavity to serve as depots for cancer slow-release immunotherapy. Sustained release of CPMV within the intraperitoneal space alleviates the need for repeated dosing and we demonstrated efficacy against ovarian cancer in a metastatic mouse model.
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Triggering lysosome-regulated immunogenic cell death (ICD, e.g., pyroptosis and necroptosis) with nanomedicines is an emerging approach for turning an "immune-cold" tumor "hot"-a key challenge faced by cancer immunotherapies. Proton sponge such as high-molecular-weight branched polyethylenimine (PEI) is excellent at rupturing lysosomes, but its therapeutic application is hindered by uncontrollable toxicity due to fixed charge density and poor understanding of resulted cell death mechanism. Here, a series of proton sponge nano-assemblies (PSNAs) with self-assembly controllable surface charge density and cell cytotoxicity are created. Such PSNAs are constructed via low-molecular-weight branched PEI covalently bound to self-assembling peptides carrying tetraphenylethene pyridinium (PyTPE, an aggregation-induced emission-based luminogen). Assembly of PEI assisted by the self-assembling peptide-PyTPE leads to enhanced surface positive charges and cell cytotoxicity of PSNA. The self-assembly tendency of PSNAs is further optimized by tuning hydrophilic and hydrophobic components within the peptide, thus resulting in the PSNA with the highest fluorescence, positive surface charge density, cell uptake, and cancer cell cytotoxicity. Systematic cell death mechanistic studies reveal that the lysosome rupturing-regulated pyroptosis and necroptosis are at least two causes of cell death. Tumor cells undergoing PSNA-triggered ICD activate immune cells, suggesting the great potential of PSNAs to trigger anticancer immunity.
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Muerte Celular Inmunogénica , Lisosomas , Péptidos , Polietileneimina , Protones , Lisosomas/metabolismo , Humanos , Péptidos/química , Muerte Celular Inmunogénica/efectos de los fármacos , Polietileneimina/química , Línea Celular Tumoral , Neoplasias/patología , Nanopartículas/química , Nanoestructuras/química , Supervivencia Celular/efectos de los fármacosRESUMEN
Our lab demonstrated that intratumoral Cowpea mosaic virus (CPMV) is a potent antitumor immunotherapy when used as in situ vaccine. As we pave the way for human clinical translation, formulation chemistry needs to be optimized for long-term storage of the drug candidate. In this work, CPMV was nanoengineered with Pluronic F127 to realize liquid and gel formulations which mitigate structural changes and RNA release during long-term storage. We evaluated the CPMV-F127 formulations for their stability and biological activity through a combination of in vitro assays and efficacy in vivo using a B16F10 murine melanoma model. Results demonstrate that both F127 liquid and gel formulations preserve CPMV structure and function following extended periods of thermal incubation at 4°C, 25°C, and 37°C. Heat-incubated CPMV without formulation resulted in structural changes and inferior in vivo efficacy. In stark contrast, in vivo efficacy was preserved when CPMV was formulated and protected with the F127 "nanoarmor."
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Many virus-like particles (VLPs) have good chemical, thermal, and mechanical stabilities compared to those of other biologics. However, their stability needs to be improved for the commercialization and use in translation of VLP-based materials. We developed an endoskeleton-armored strategy for enhancing VLP stability. Specifically, the VLPs of physalis mottle virus (PhMV) and Qß were used to demonstrate this concept. We built an internal polymer "backbone" using a maleimide-PEG15-maleimide cross-linker to covalently interlink viral coat proteins inside the capsid cavity, while the native VLPs are held together by only noncovalent bonding between subunits. Endoskeleton-armored VLPs exhibited significantly improved thermal stability (95 °C for 15 min), increased resistance to denaturants (i.e., surfactants, pHs, chemical denaturants, and organic solvents), and enhanced mechanical performance. Single-molecule force spectroscopy demonstrated a 6-fold increase in rupture distance and a 1.9-fold increase in rupture force of endoskeleton-armored PhMV. Overall, this endoskeleton-armored strategy provides more opportunities for the development and applications of materials.
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Proteínas de la Cápside , Cápside , Proteínas de la Cápside/química , Cápside/química , Maleimidas/análisisRESUMEN
PEGylation is the gold standard in protein-polymer conjugation, improving circulation half-life of biologics while mitigating the immune response to a foreign substance. However, preexisting anti-PEG antibodies in healthy humans are becoming increasingly prevalent and elicitation of anti-PEG antibodies when patients are administered with PEGylated therapeutics challenges their safety profile. In the current study, two distinct amine-reactive poly(oxanorbornene) (PONB) imide-based water-soluble block co-polymers are synthesized using ring-opening metathesis polymerization (ROMP). The synthesized block-copolymers include PEG-based PONB-PEG and sulfobetaine-based PONB-Zwit. The polymers are then covalently conjugated to amine residues of lysozyme (Lyz) and urate oxidase (UO) using a grafting-to bioconjugation technique. Both Lyz-PONB and UO-PONB conjugates retained significant bioactivities after bioconjugation. Immune recognition studies of UO-PONB conjugates indicated a comparable lowering of protein immunogenicity when compared to PEGylated UO. PEG-specific immune recognition is negligible for UO-PONB-Zwit conjugates, as expected. These polymers provide a new alternative for PEG-based systems that retain high levels of activity for the biologic while showing improved immune recognition profiles.
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Polietilenglicoles , Proteínas , Humanos , Polietilenglicoles/química , Polimerizacion , Proteínas/química , Polímeros/química , AminasRESUMEN
Atherosclerosis is an immune-mediated chronic inflammatory disease that leads to the development of fatty plaques in the arterial walls, ultimately increasing the risk of thrombosis, stroke, and myocardial infarction. The immune response in this complex disease is both atheroprotective and pro-atherogenic, involving both innate and adaptive immunity. Current treatments include the adjustment of lifestyle factors, cholesterol-lowering drugs such as statins, and immunotherapy, whereas vaccine development has received comparatively little attention. In this review, we discuss the potential of antigen-specific vaccination as a preventative approach based on more than 20 years of research and innovation. Vaccination targets include proteins that are more abundant in atherosclerotic patients, such as oxidized low-density lipoprotein (LDL), apolipoprotein B-100, proprotein convertase subtilisin/kexin type-9 serine protease (PCSK9), cholesteryl ester transfer protein (CETP), and heat shock proteins HSP60 and HSP65. Immunization with such proteins or their peptide epitopes has been shown to induce T-cell activation, produce antigen-specific antibodies, reduce the size of atherosclerotic lesions, and/or reduce serum cholesterol levels. Vaccination against atherosclerosis therefore offers a new strategy to address the burden on healthcare systems caused by cardiovascular disease, the leading cause of death worldwide.