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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 , Folhas de Planta , Norbornanos/química , Folhas de Planta/química , Polimerização , Polímeros/química , PlásticosRESUMO
The field of hybrid engineered living materials seeks to pair living organisms with synthetic materials to generate biocomposite materials with augmented function since living systems can provide highly-programmable and complex behavior. Engineered living materials have typically been fabricated using techniques in benign aqueous environments, limiting their application. In this work, biocomposite fabrication is demonstrated in which spores from polymer-degrading bacteria are incorporated into a thermoplastic polyurethane using high-temperature melt extrusion. Bacteria are engineered using adaptive laboratory evolution to improve their heat tolerance to ensure nearly complete cell survivability during manufacturing at 135 °C. Furthermore, the overall tensile properties of spore-filled thermoplastic polyurethanes are substantially improved, resulting in a significant improvement in toughness. The biocomposites facilitate disintegration in compost in the absence of a microbe-rich environment. Finally, embedded spores demonstrate a rationally programmed function, expressing green fluorescent protein. This research provides a scalable method to fabricate advanced biocomposite materials in industrially-compatible processes.
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Materiais Biocompatíveis , Poliuretanos , Esporos Bacterianos , Poliuretanos/química , Materiais Biocompatíveis/química , Resistência à Tração , Temperatura Alta , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genéticaRESUMO
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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Praguicidas , Vírus do Mosaico do Tabaco , Tobamovirus , Ecossistema , Praguicidas/metabolismo , Solo , VírionRESUMO
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 Oxidase , Humanos , Polimerização , ProteínasRESUMO
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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Polietilenoglicóis , Proteínas , Humanos , Polietilenoglicóis/química , Polimerização , Proteínas/química , Polímeros/química , AminasRESUMO
The field of engineered living materials lies at the intersection of materials science and synthetic biology with the aim of developing materials that can sense and respond to the environment. In this study, we use 3D printing to fabricate a cyanobacterial biocomposite material capable of producing multiple functional outputs in response to an external chemical stimulus and demonstrate the advantages of utilizing additive manufacturing techniques in controlling the shape of the fabricated photosynthetic material. As an initial proof-of-concept, a synthetic riboswitch is used to regulate the expression of a yellow fluorescent protein reporter in Synechococcus elongatus PCC 7942 within a hydrogel matrix. Subsequently, a strain of S. elongatus is engineered to produce an oxidative laccase enzyme; when printed within a hydrogel matrix the responsive biomaterial can decolorize a common textile dye pollutant, indigo carmine, potentially serving as a tool in environmental bioremediation. Finally, cells are engineered for inducible cell death to eliminate their presence once their activity is no longer required, which is an important function for biocontainment and minimizing environmental impact. By integrating genetically engineered stimuli-responsive cyanobacteria in volumetric 3D-printed designs, we demonstrate programmable photosynthetic biocomposite materials capable of producing functional outputs including, but not limited to, bioremediation.
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Synechococcus , Synechococcus/genética , Synechococcus/metabolismo , Fotossíntese , Biologia Sintética/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Engenharia Metabólica/métodos , Hidrogéis/metabolismoRESUMO
Spherical nanoparticles (SNPs) from tobacco mild green mosaic virus (TMGMV) were developed and characterized, and their application for agrochemical delivery was demonstrated. Specifically, we set out to develop a platform for pesticide delivery targeting nematodes in the rhizosphere. SNPs were obtained by thermal shape-switching of the TMGMV. We demonstrated that cargo can be loaded into the SNPs during thermal shape-switching, enabling the one-pot synthesis of functionalized nanocarriers. Cyanine 5 and ivermectin were encapsulated into SNPs to achieve 10% mass loading. SNPs demonstrated good mobility and soil retention slightly higher than that of TMGMV rods. Ivermectin delivery to Caenorhabditis elegans using SNPs was determined after passing the formulations through soil. Using a gel burrowing assay, we demonstrate the potent efficacy of SNP-delivered ivermectin against nematodes. Like many pesticides, free ivermectin is adsorbed in the soil and did not show efficacy. The SNP nanotechnology offers good soil mobility and a platform technology for pesticide delivery to the rhizosphere.
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Nanopartículas , Praguicidas , Vírus do Mosaico do Tabaco , Animais , Vírus do Mosaico do Tabaco/química , Ivermectina/farmacologia , Nanopartículas/química , Praguicidas/farmacologia , Caenorhabditis elegans , SoloRESUMO
Single dose slow-release vaccines herald a new era in vaccine administration. An ideal device for slow-release vaccine delivery would be minimally invasive and self-administered, making these approaches an attractive alternative for mass vaccination programs, particularly during the time of a pandemic. In this review article, we discuss the latest advances in this field, specifically for prophylactic vaccines able to prevent infectious diseases. Recent studies have found that slow-release vaccines elicit better immune responses and often do not require cold chain transportation and storage, thus drastically reducing the cost, streamlining distribution, and improving efficacy. This promise has attracted significant attention, especially when poor patient compliance of the standard multidose vaccine regimes is considered. Single dose slow-release vaccines are the next generation of vaccine tools that could overcome most of the shortcomings of present vaccination programs and be the next platform technology to combat future pandemics. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Implantable Materials and Surgical Technologies > Nanomaterials and Implants Biology-Inspired Nanomaterials > Protein and Virus-Based Structures.
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Nanoestruturas , Vacinas , Humanos , Vacinação , Sistemas de Liberação de Medicamentos , Descoberta de DrogasRESUMO
The standard therapy for cardiovascular disease (CVD) is the administration of statins to reduce plasma cholesterol levels, but this requires lifelong treatment. We developed a CVD vaccine candidate that targets the pro-inflammatory mediator calprotectin by eliciting antibodies against the S100A9 protein. The vaccine, based on bacteriophage Qß virus-like particles (VLPs) displaying S100A9 peptide epitopes, was formulated as a slow-release PLGA:VLP implant by hot-melt extrusion. The single-dose implant elicited S100A9-specific antibody titers comparable to a three-dose injection schedule with soluble VLPs. In an animal model of CVD (ApoE-/- mice fed on a high-fat diet), the implant reduced serum levels of calprotectin, IL-1ß, IL-6 and MCP-1, resulting in less severe aortic lesions. This novel implant was therefore able to attenuate atherosclerosis over a sustained period and offers a novel and promising strategy to replace the repetitive administration of statins for the treatment of CVD.
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Recent developments of highly expandable foaming pre-polymer resins for lithographic additive manufacturing have allowed for the creation of structures larger than a printer's build envelope. To fully utilize the capabilities of this technology, the mechanical properties of these foams must be improved. This manuscript presents one method for strengthening these lightweight polymeric structures via aerosol spray application of a high-strength, low-viscosity photocurable coating. This method is free from the reliance on often complex, large, or bulky on-site equipment ordinarily required by conventional high-strength spray coating. The newly formulated photocurable resin can be applied using an ordinary cordless paint sprayer and cured using sunlight in less than a minute, enabling the rapid production of large, load-bearing structures from a small volume of feedstock and low-cost portable equipment. A comprehensive screening process for resin formulations, detailed mechanical compression and tensile analysis of coated polymer structures, and an applied technical demonstration of the technology are described. The photocurable coating described herein greatly strengthens porous polymeric structures using a method that can be easily implemented.
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Blood loss causes an estimated 1.9 million deaths per year globally, making new methods to stop bleeding and promote clot formation immediately following injury paramount. The fabrication of functional hemostatic materials has the potential to save countless lives by limiting bleeding and promoting clot formation following an injury. This work describes the melt manufacturing of poly(ε-caprolactone) nanofibers and their chemical functionalization to produce highly scalable materials with enhanced blood clotting properties. The nanofibers are manufactured using a high throughput melt coextrusion method. Once isolated, the nanofibers are functionalized with polymers that promote blood clotting through surface-initiated atom transfer radical polymerization. The functional nanofibers described herein speed up the coagulation cascade and produce more robust blood clots, allowing for the potential use of these functional nonwoven mats as advanced bandages.
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Hemostáticos , Nanofibras , Trombose , Humanos , Nanofibras/química , Coagulação Sanguínea , Hemostáticos/química , Bandagens , HemorragiaRESUMO
Prophylactic vaccines capable of preventing human papillomavirus (HPV) infections are still inaccessible to a vast majority of the global population due to their high cost and challenges related to multiple administrations performed in a medical setting. In an effort to improve distribution and administration, we have developed dissolvable microneedles loaded with a thermally stable HPV vaccine candidate consisting of Qß virus-like particles (VLPs) displaying a highly conserved epitope from the L2 protein of HPV (Qß-HPV). Polymeric microneedle delivery of Qß-HPV produces similar amounts of anti-HPV16 L2 IgG antibodies compared to traditional subcutaneous injection while delivering a much smaller amount of intradermal dose. However, a dose sparing effect was found. Furthermore, immunization yielded neutralizing antibody responses in a HPV pseudovirus assay. The vaccine candidate was confirmed to be stable at room temperature after storage for several months, potentially mitigating many of the challenges associated with cold-chain distribution. The ease of self-administration and minimal invasiveness of such microneedle patch vaccines may enable wide-scale distribution of the HPV vaccine and lead to higher patient compliance. The Qß VLP and its delivery technology is a plug-and-play system that could serve as a universal platform with a broad range of applications. Qß VLPs may be stockpiled for conjugation to a wide range of epitopes, which are then packaged and delivered directly to the patient via noninvasive microneedle patches. Such a system paves the way for rapid distribution and self-administration of vaccines.
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Infecções por Papillomavirus , Vacinas contra Papillomavirus , Vacinas de Partículas Semelhantes a Vírus , Animais , Anticorpos Antivirais , Proteínas do Capsídeo , Epitopos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/uso terapêuticoRESUMO
Protein-polymer conjugates (PPCs) improve therapeutic efficacy of proteins and have been widely used for the treatment of various diseases such as cancer, diabetes, and hepatitis. PEGylation is considered as the "gold standard" in bioconjugation, although in practice its clinical applications are becoming limited because of extensive evidence of immunogenicity induced by pre-existing anti-PEG antibodies in patients. Here, optimized reaction conditions for living aqueous grafting-from ring-opening metathesis polymerization (ROMP) are utilized to synthesize water-soluble polynorbornene (PNB)-based PPCs of lysozyme (Lyz-PPCs) and bacteriophage Qß (Qß-PPCs) as PEG alternatives. Lyz-PPCs retain nearly 100% bioactivity and Qß-PPCs exhibit up to 35% decrease in protein immunogenicity. Qß-PPCs derived from NB-PEG show no reduction in recognition by anti-PEG antibodies while Qß-PPCs derived from NB-Zwit show >95% reduction as compared with Qß-PEG. This work demonstrates a new method for PPC synthesis and the utility of grafting from PPCs to evade immune recognition.
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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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Proteinaceous nanoparticles can be used to deliver large payloads of active ingredients, which is advantageous in medicine and agriculture. However, the conjugation of hydrophobic ligands to hydrophilic nanocarriers such as plant viral nanoparticles (plant VNPs) can result in aggregation by reducing overall solubility. Given the benefits of hydrophilic nanocarrier platforms for targeted delivery and multivalent ligand display, coupled with the versatility of hydrophobic drugs, contrast agents, and peptides, this is an issue that must be addressed to realize their full potential. Here, we report two preincubation strategies that use a Pluronic F127 polymer scaffold to prevent the aggregation of conjugated plant VNPs: a plant VNP-polymer precoat (COAT) and an active ingredient formulation combined with a plant VNP-polymer precoat (FORMCOAT). The broad applications of these modified conjugation strategies were highlighted by testing their compatibility with three types of bioconjugation chemistry: N-hydroxysuccinimide ester-amine coupling, maleimide-thiol coupling, and copper(I)-catalyzed azide-alkyne cycloaddition (click chemistry). The COAT and FORMCOAT strategies promoted efficient bioconjugation and prevented the aggregation that accompanies conventional bioconjugation methods, thus improving the stability, homogeneity, and translational potential of plant VNP conjugates in medicine and agriculture.
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Materiais Biocompatíveis/química , Nanopartículas/química , Poloxâmero/química , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Teste de Materiais , Estrutura Molecular , Tamanho da PartículaRESUMO
Progress in the field of soft devices-i.e., haptics, robotics, and human-machine interfaces (HRHMIs)-has its basis in the science of polymeric materials and chemical synthesis. However, in examining the relevant literature, we find that most developments have been enabled by off-the-shelf materials used either alone or as components of physical blends and composites. In this Progress Report, we take the position that a greater awareness of the capabilities of synthetic chemistry will accelerate the capabilities of HRHMIs. Conversely, an awareness of the applications sought by engineers working in this area may spark the development of new molecular designs and synthetic methodologies by chemists. We highlight several applications of active, stimuli-responsive polymers, which have demonstrated or shown potential use in HRHMIs. These materials share the fact that they are products of state-of-the-art synthetic techniques. The Progress Report is thus organized by the chemistry by which the materials were synthesized, including controlled radical polymerization, metal-mediated cross-coupling polymerization, ring-opening polymerization, various strategies for crosslinking, and hybrid approaches. These methods can afford polymers with multiple properties (i.e. conductivity, stimuli-responsiveness, self-healing and degradable abilities, biocompatibility, adhesiveness, and mechanical robustness) that are of great interest to scientists and engineers concerned with soft devices for human interaction.
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The development of vaccines against coronaviruses has focused on the spike (S) protein, which is required for the recognition of host-cell receptors and thus elicits neutralizing antibodies. Targeting conserved epitopes on the S protein offers the potential for pan-beta-coronavirus vaccines that could prevent future pandemics. We displayed five B-cell epitopes, originally identified in the convalescent sera from recovered severe acute respiratory syndrome (SARS) patients, on the surface of the cowpea mosaic virus (CPMV) and evaluated these formulations as vaccines. Prime-boost immunization of mice with three of these candidate vaccines, CPMV-988, CPMV-1173, and CPMV-1209, elicited high antibody titers that neutralized the severe acute respiratory syndrome coronavirus (SARS-CoV) in vitro and showed an early Th1-biased profile (2-4 weeks) transitioning to a slightly Th2-biased profile just after the second boost (6 weeks). A pentavalent slow-release implant comprising all five peptides displayed on the CPMV elicited anti-S protein and epitope-specific antibody titers, albeit at a lower magnitude compared to the soluble formulations. While the CPMV remained intact when released from the PLGA implants, processing results in loss of RNA, which acts as an adjuvant. Loss of RNA may be a reason for the lower efficacy of the implants. Finally, although the three epitopes (988, 1173, and 1209) that were found to be neutralizing the SARS-CoV were 100% identical to the SARS-CoV-2, none of the vaccine candidates neutralized the SARS-CoV-2 in vitro suggesting differences in the natural epitope perhaps caused by conformational changes or the presence of N-linked glycans. While a cross-protective vaccine candidate was not developed, a multivalent SARS vaccine was developed. The technology discussed here is a versatile vaccination platform that can be pivoted toward other diseases and applications that are not limited to infectious diseases.
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COVID-19 , Comovirus , Nanopartículas , Vacinas , Animais , COVID-19/terapia , Comovirus/genética , Epitopos de Linfócito B , Humanos , Imunização Passiva , Camundongos , Peptídeos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Soroterapia para COVID-19RESUMO
Cardiovascular disease is the number one cause of death globally. Lowering cholesterol levels in plasma is the mainstay therapy; however lifelong treatment and adverse effects call for improved therapeutic interventions. We developed a trivalent vaccine candidate targeting proprotein convertase subtilisin/kexin-9 (PCSK9), apolipoprotein B (ApoB), and cholesteryl ester transfer protein (CETP). Vaccine candidates were developed using bacteriophage Qß-based virus-like particles (VLPs) displaying antigens of PCKS9, ApoB, and CETP, respectively. Vaccine candidate mixtures were formulated as slow-release PLGA:VLP implants using hot-melt extrusion. The delivery of the trivalent vaccine candidate via the implant produced antibodies against the cholesterol checkpoint proteins at levels comparable to a three-dose injection schedule with soluble mixtures. The reduction in PCSK9 and ApoB levels in plasma, inhibition of CETP (in vitro), and total plasma cholesterol decrease was achieved. All-together, we present a platform technology for a single-dose multi-target vaccination platform targeting cholesterol checkpoint proteins.
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The COVID-19 pandemic highlights the need for platform technologies enabling rapid development of vaccines for emerging viral diseases. The current vaccines target the SARS-CoV-2 spike (S) protein and thus far have shown tremendous efficacy. However, the need for cold-chain distribution, a prime-boost administration schedule, and the emergence of variants of concern (VOCs) call for diligence in novel SARS-CoV-2 vaccine approaches. We studied 13 peptide epitopes from SARS-CoV-2 and identified three neutralizing epitopes that are highly conserved among the VOCs. Monovalent and trivalent COVID-19 vaccine candidates were formulated by chemical conjugation of the peptide epitopes to cowpea mosaic virus (CPMV) nanoparticles and virus-like particles (VLPs) derived from bacteriophage Qß. Efficacy of this approach was validated first using soluble vaccine candidates as solo or trivalent mixtures and subcutaneous prime-boost injection. The high thermal stability of our vaccine candidates allowed for formulation into single-dose injectable slow-release polymer implants, manufactured by melt extrusion, as well as microneedle (MN) patches, obtained through casting into micromolds, for prime-boost self-administration. Immunization of mice yielded high titers of antibodies against the target epitope and S protein, and data confirms that antibodies block receptor binding and neutralize SARS-CoV and SARS-CoV-2 against infection of human cells. We present a nanotechnology vaccine platform that is stable outside the cold-chain and can be formulated into delivery devices enabling single administration or self-administration. CPMV or Qß VLPs could be stockpiled, and epitopes exchanged to target new mutants or emergent diseases as the need arises.
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Vacinas contra COVID-19/metabolismo , COVID-19/epidemiologia , COVID-19/prevenção & controle , Preparações de Ação Retardada/química , SARS-CoV-2/metabolismo , Vacinas de Subunidades Antigênicas/metabolismo , Animais , Comovirus , Simulação por Computador , Composição de Medicamentos , Epitopos/química , Temperatura Alta , Humanos , Masculino , Camundongos Endogâmicos BALB C , Nanopartículas/química , Peptídeos/química , Vacinação , Vacinas de Partículas Semelhantes a Vírus/químicaRESUMO
Prostate-specific membrane antigen (PSMA) is a membrane-bound protein that is preferentially expressed in the prostate gland and induced in many prostate cancers, making it an important target for new diagnostics and therapeutics. To improve the efficacy of nanoparticle formulations for the imaging and/or eradication of prostate cancer, we synthesized the PSMA-binding glutamic acid derivative DUPA and conjugated it to the external surface of tobacco mosaic virus (TMV) particles. DUPA-targeted TMV was subsequently loaded with the antineoplastic agent mitoxantrone (MTO) or conjugated internally with the fluorescent dye cyanine 5 (Cy5). We found that TMV particles could be efficiently decorated with DUPA and loaded with MTO or Cy5 while maintaining structural integrity. DUPA-targeted TMV particles were able to bind more efficiently to the surface of PSMA+ LNCaP cells compared to non-targeted TMV; but there was little difference in binding efficiency between targeted and untargeted TMV when we tested PSMA- PC3 cells (both cell lines are prostate cancer cell lines). DUPA-targeted TMV particles were internalized by LNCaP cells enabling drug delivery. Finally, we loaded the DUPA-targeted TMV particles and untargeted control particles with MTO to test their cytotoxicity against LNCaP cells in vitro. The cytotoxicity of the TMV-MTO particles (IC50 = 10.2 nM) did not differ significantly from that of soluble MTO at an equivalent dose (IC50 = 12.5 nM) but the targeted particles (TMV-DUPA-MTO) were much more potent (IC50 = 2.80 nM). The threefold increase in cytotoxicity conferred by the DUPA ligand suggests that MTO-loaded, DUPA-coated TMV particles are promising as a therapeutic strategy for PSMA+ prostate cancer and should be advanced to preclinical testing in mouse models of prostate cancer.