Viral nanoparticle vaccines against S100A9 reduce lung tumor seeding and metastasis.

Metastatic cancer accounts for 90% of all cancer-related deaths and continues to be one of the toughest challenges in cancer treatment. A growing body of data indicates that S100A9, a major regulator of inflammation, plays a central role in cancer progression and metastasis, particularly in the lungs, where S100A9 forms a premetastatic niche. Thus, we developed a vaccine against S100A9 derived from plant viruses and virus-like particles. Using multiple tumor mouse models, we demonstrate the effectiveness of the S100A9 vaccine candidates in preventing tumor seeding within the lungs and outgrowth of metastatic disease. The elicited antibodies showed high specificity toward S100A9 without cross-reactivity toward S100A8, another member of the S100A family. When tested in metastatic mouse models of breast cancer and melanoma, the vaccines significantly reduced lung tumor nodules after intravenous challenge or postsurgical removal of the primary tumor. Mechanistically, the vaccines reduce the levels of S100A9 within the lungs and sera, thereby increasing the expression of immunostimulatory cytokines with antitumor function [(interleukin) IL-12 and interferonγ] while reducing levels of immunosuppressive cytokines (IL-10 and transforming growth factorß). This also correlated with decreased myeloid-derived suppressor cell populations within the lungs. This work has wide-ranging impact, as S100A9 is overexpressed in multiple cancers and linked with poor prognosis in cancer patients. The data presented lay the foundation for the development of therapies and vaccines targeting S100A9 to prevent metastasis.

Virus-like Particles Armored by an Endoskeleton.

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.

DNA Delivery by Virus-Like Nanocarriers in Plant Cells.

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.

Synergistic combination therapy using cowpea mosaic virus intratumoral immunotherapy and Lag-3 checkpoint blockade.

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.

Biochemical and nanotechnological approaches to combat phytoparasitic nematodes.

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.

In Vivo Delivery of Spherical and Cylindrical In Vitro Reconstituted Virus-like Particles Containing the Same Self-Amplifying mRNA.

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.

Delivery of Nematicides Using TMGMV-Derived Spherical Nanoparticles.

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.

TLR Agonists Delivered by Plant Virus and Bacteriophage Nanoparticles for Cancer Immunotherapy.

Toll-like receptors (TLRs) are promising targets in cancer immunotherapy due to their role in activating the immune system; therefore, various small-molecule TLR agonists have been tested in clinical applications. However, the clinical use of TLR agonists is hindered by their non-specific side effects and poor pharmacokinetics. To overcome these limitations, we used plant virus nanoparticles (VNPs) and bacteriophage virus-like particles (VLPs) as drug delivery systems. We conjugated TLR3 or TLR7 agonists to cowpea mosaic virus (CPMV) VNPs, cowpea chlorotic mottle virus (CCMV) VNPs, and bacteriophage Qß VLPs. The conjugation of TLR7 agonist, 2-methoxyethoxy-8-oxo-9-(4-carboxybenzyl)adenine (1V209), resulted in the potent activation of immune cells and promoted the production of pro-inflammatory cytokine interleukin 6. We found that 1V209 conjugated to CPMV, CCMV, and Qß reduced tumor growth in vivo and prolonged the survival of mice compared to those treated with free 1V209 or a simple admixture of 1V209 and viral particles. Nucleic acid-based TLR3 agonist, polyinosinic acid with polycytidylic acid (poly(I:C)), was also delivered by CPMV VNPs, resulting in enhanced mice survival. All our data suggest that coupling and co-delivery are required to enhance the anti-tumor efficacy of TLR agonists and simple mixing of the VLPs with the agonists does not confer a survival benefit. The delivery of 1V209 or poly(I:C) conjugated to VNPs/VLPs probably enhances their efficacy due to the multivalent presentation, prolongation of tumor residence time, and targeting of the innate immune cells mediated by the VNP/VLP carrier.

Development of a Modular NTA:His Tag Viral Vaccine for Co-delivery of Antigen and Adjuvant.

The SARS-CoV-2 pandemic has highlighted the need for vaccines that are effective, but quickly produced. Of note, vaccines with plug-and-play capabilities that co-deliver antigen and adjuvant to the same cell have shown remarkable success. Our approach of utilizing a nitrilotriacetic acid (NTA) histidine (His)-tag chemistry with viral adjuvants incorporates both of these characteristics: plug-and-play and co-delivery. We specifically utilize the cowpea mosaic virus (CPMV) and the virus-like particles from bacteriophage Qß as adjuvants and bind the model antigen ovalbumin (OVA). Successful binding of the antigen to the adjuvant/carrier was verified by SDS-PAGE, western blot, and ELISA. Immunization in C57BL/6J mice demonstrates that with Qß - but not CPMV - there is an improved antibody response against the target antigen using the Qß-NiNTA:His-OVA versus a simple admixture of antigen and adjuvant. Antibody isotyping also shows that formulation of the vaccines can alter T helper biases; while the Qß-NiNTA:His-OVA particle produces a balanced Th1/Th2 bias the admixture was strongly Th2. In a mouse model of B16F10-OVA, we further demonstrate improved survival and slower tumor growth in the vaccine groups compared to controls. The NiNTA:His chemistry demonstrates potential for rapid development of future generation vaccines enabling plug-and-play capabilities with effectiveness boosted by co-delivery to the same cell.

Physalis Mottle Virus-Like Nanocarriers with Expanded Internal Loading Capacity.

An ongoing challenge in precision medicine is the efficient delivery of therapeutics to tissues/organs of interest. Nanoparticle delivery systems have the potential to overcome traditional limitations of drug and gene delivery through improved pharmacokinetics, tissue targeting, and stability of encapsulated cargo. Physalis mottle virus (PhMV)-like nanoparticles are a promising nanocarrier platform which can be chemically targeted on the exterior and interior surfaces through reactive amino acids. Cargo-loading to the internal cavity is achieved with thiol-reactive small molecules. However, the internal loading capacity of these nanoparticles is limited by the presence of a single reactive cysteine (C75) per coat protein with low inherent reactivity. Here, we use structure-based design to engineer cysteine-added mutants of PhMV VLPs that display increased reactivity toward thiol-reactive small molecules. Specifically, the A31C and S137C mutants show a greater than 10-fold increased rate of reactivity towards thiol-reactive small molecules, and PhMV Cys1 (A31C), PhMV Cys2 (S137C), and PhMV Cys1+2 (double mutant) VLPs display up to three-fold increased internal loading of the small molecule chemotherapeutics aldoxorubicin and vcMMAE and up to four-fold increased internal loading of the MRI imaging reagent DOTA(Gd). These results further improve upon a promising plant virus-based nanocarrier system for use in targeted delivery of small-molecule drugs and imaging reagents in vivo.

The Potency of Cowpea Mosaic Virus Particles for Cancer In Situ Vaccination Is Unaffected by the Specific Encapsidated Viral RNA.

Plant virus nanoparticles can be used as drug carriers, imaging reagents, vaccine carriers, and immune adjuvants in the formulation of intratumoral in situ cancer vaccines. One example is the cowpea mosaic virus (CPMV), a nonenveloped virus with a bipartite positive-strand RNA genome with each RNA packaged separately into identical protein capsids. Based on differences in their densities, the components carrying RNA-1 (6 kb) denoted as the bottom (B) component or carrying RNA-2 (3.5 kb) denoted as the middle (M) component can be separated from each other and from a top (T) component, which is devoid of any RNA. Previous preclinical mouse studies and canine cancer trials used mixed populations of CPMV (containing B, M, and T components), so it is unclear whether the particle types differ in their efficacies. It is known that the CPMV RNA genome contributes to immunostimulation by activation of TLR7. To determine whether the two RNA genomes that have different sizes and unrelated sequences cause different immune stimulation, we compared the therapeutic efficacies of B and M components and unfractionated CPMV in vitro and in mouse cancer models. We found that separated B and M particles behaved similarly to the mixed CPMV, activating innate immune cells to induce the secretion of pro-inflammatory cytokines such as IFNα, IFNγ, IL-6, and IL-12, while inhibiting immunosuppressive cytokines such as TGF-ß and IL-10. In murine models of melanoma and colon cancer, the mixed and separated CPMV particles all significantly reduced tumor growth and prolonged survival with no significant difference. This shows that the specific RNA genomes similarly stimulate the immune system even though B particles have 40% more RNA than M particles; each CPMV particle type can be used as an effective adjuvant against cancer with the same efficacy as native mixed CPMV. From a translational point of view, the use of either B or M component vs the mixed CPMV formulation offers the advantage that separated B or M alone is noninfectious toward plants and thus provides agronomic safety.

Inactivated Cowpea Mosaic Virus for In Situ Vaccination: Differential Efficacy of Formalin vs UV-Inactivated Formulations.

Cowpea mosaic virus (CPMV) has been developed as a promising nanoplatform technology for cancer immunotherapy; when applied as in situ vaccine, CPMV exhibits potent, systemic, and durable efficacy. While CPMV is not infectious to mammals, it is infectious to legumes; therefore, agronomic safety needs to be addressed to broaden the translational application of CPMV. RNA-containing formulations are preferred over RNA-free virus-like particles because the RNA and protein, each, contribute to CPMV's potent antitumor efficacy. We have previously optimized inactivation methods to develop CPMV that contains RNA but is not infectious to plants. We established that inactivated CPMV has reduced efficacy compared to untreated, native CPMV. However, a systematic comparison between native CPMV and different inactivated forms of CPMV was not done. Therefore, in this study, we directly compared the therapeutic efficacies and mechanisms of immune activation of CPMV, ultraviolet- (UV-), and formalin (Form)-inactivated CPMV to explain the differential efficacies. In a B16F10 melanoma mouse tumor model, Form-CPMV suppressed the tumor growth with prolonged survival (there were no statistical differences comparing CPMV and Form-CPMV). In comparison, UV-CPMV inhibited tumor growth significantly but not as well as Form-CPMV or CPMV. The reduced therapeutic efficacy of UV-CPMV is explained by the degree of cross-linking and aggregated state of the RNA, which renders it inaccessible for sensing by Toll-like receptor (TLR) 7/8 to activate immune responses. The mechanistic studies showed that the highly aggregated state of UV-CPMV inhibited TLR7 signaling more so than for the Form-CPMV formulation, reducing the secretion of interleukin-6 (IL-6) and interferon-α (IFN-α), cytokines associated with TLR7 signaling. These findings support the translational development of Form-CPMV as a noninfectious immunotherapeutic agent.

In Vitro and Ex Planta Gold-Bonded and Gold-Mineralized Tobacco Mosaic Virus.

Biotemplated mineralization is a promising and ecofriendly approach to manufacture metal nanoparticles and composites with precise size control. Plant viruses are suitable templates for biomineralization because they are chemically robust and highly scalable through molecular farming. Here, we report a gold-nanoparticle-coated tobacco mosaic virus (TMV) synthesized in a test tube or in plant extracts making use of a TMV displaying a gold-binding peptide (GBP). The methods developed are a step toward engineered living materials, where gold nanowires could be formed in plant tissues for sensing or energy harvest applications.

Treatment of Hepatocellular Carcinoma by Multimodal In Situ Vaccination Using Cryoablation and a Plant Virus Immunostimulant.

PURPOSE: To test the hypothesis that cryoablation combined with intratumoral immunomodulating nanoparticles from cowpea mosaic virus (CPMV) as an in situ vaccination approach induces systemic antitumoral immunity in a murine model of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Mice with bilateral, subcutaneous RIL-175 cell-derived HCCs were randomized to 4 groups: (a) phosphate-buffered saline (control), (b) cryoablation only (Cryo), (c) CPMV-treated only (CPMV), and (d) cryoablation plus CPMV-treated (Cryo + CPMV) (N = 11-14 per group). Intratumoral CPMV was administered every 3 days for 4 doses, with cryoablation performed on the third day. Contralateral tumors were monitored. Tumor growth and systemic chemokine/cytokine levels were measured. A subset of tumors and spleens were harvested for immunohistochemistry (IHC) and flow cytometry. One- or 2-way analysis of variance was performed for statistical comparisons. A P value of <.05 was used as the threshold for statistical significance. RESULTS: At 2 weeks after treatment, the Cryo and CPMV groups, alone or combined, outperformed the control group in the treated tumor; however, the Cryo + CPMV group showed the strongest reduction and lowest variance (1.6-fold ± 0.9 vs 6.3-fold ± 0.5, P < .0001). For the untreated tumor, only Cryo + CPMV significantly reduced tumor growth compared with control (9.2-fold ± 0.9 vs 17.8-fold ± 2.1, P = .01). The Cryo + CPMV group exhibited a transient increase in interleukin-10 and persistently decreased CXCL1. Flow cytometry revealed natural killer cell enrichment in the untreated tumor and increased PD-1 expression in the spleen. Tumor-infiltrating lymphocytes increased in Cryo + CPMV-treated tumors by IHC. CONCLUSIONS: Cryoablation and intratumoral CPMV, alone or combined, demonstrated potent efficacy against treated HCC tumors; however, only cryoablation combined with CPMV slowed the growth of untreated tumors, consistent with an abscopal effect.

Cowpea Mosaic Virus and Natural Killer Cell Agonism for In Situ Cancer Vaccination.

We have previously shown the plant virus Cowpea mosaic virus (CPMV) to be an efficacious in situ cancer vaccine, providing elimination of tumors and tumor-specific immune memory. Additionally, we have shown that CPMV recruits Natural Killer (NK) cells within the tumor microenvironment. Here we aimed to determine whether a combination of CPMV and anti-4-1BB monoclonal antibody agonist to stimulate tumor-resident and CPMV-recruited NK cells is an effective dual therapy approach to improve NK cell function and in situ cancer vaccination efficacy. Using murine models of metastatic colon carcinomatosis and intradermal melanoma, intratumorally administered CPMV + anti-4-1BB dual therapy provided a robust antitumor response, improved elimination of primary tumors, and reduced mortality compared to CPMV and anti-4-1BB monotherapies. Additionally, on tumor rechallenge there was significant delay/prevention of tumor development and improved survival, highlighting that the CPMV + anti-4-1BB dual therapy enables potent and durable antitumor efficacy.

Transcriptomics of Canine Inflammatory Mammary Cancer Treated with Empty Cowpea Mosaic Virus Implicates Neutrophils in Anti-Tumor Immunity.

Canine inflammatory mammary cancer (IMC) is a highly aggressive and lethal cancer in dogs serving as a valuable animal model for its human counterpart, inflammatory breast cancer (IBC), both lacking effective therapies. Intratumoral immunotherapy (IT-IT) with empty cowpea mosaic virus (eCPMV) nanoparticles has shown promising results, demonstrating a reduction in tumor size, longer survival rates, and improved quality of life. This study compares the transcriptomic profiles of tumor samples from female dogs with IMC receiving eCPMV IT-IT and medical therapy (MT) versus MT alone. Transcriptomic analyses, gene expression profiles, signaling pathways, and cell type profiling of immune cell populations in samples from four eCPMV-treated dogs with IMC and four dogs with IMC treated with MT were evaluated using NanoString Technologies using a canine immune-oncology panel. Comparative analyses revealed 34 differentially expressed genes between treated and untreated samples. Five genes (CXCL8, S100A9, CCL20, IL6, and PTGS2) involved in neutrophil recruitment and activation were upregulated in the treated samples, linked to the IL17-signaling pathway. Cell type profiling showed a significant increase in neutrophil populations in the tumor microenvironment after eCPMV treatment. These findings highlight the role of neutrophils in the anti-tumor response mediated by eCPMV IT-IT and suggest eCPMV as a novel therapeutic approach for IBC/IMC.

Isolation of Tobacco Mosaic Virus-Binding Peptides for Biotechnology Applications.

Tobacco mosaic virus (TMV) was the first virus to be discovered and it is now widely used as a tool for biological research and biotechnology applications. TMV particles can be decorated with functional molecules by genetic engineering or bioconjugation. However, this can destabilize the nanoparticles, and/or multiple rounds of modification may be necessary, reducing product yields and preventing the display of certain cargo molecules. To overcome these challenges, we used phage display technology and biopanning to isolate a TMV-binding peptide (TBPT25 ) with strong binding properties (IC50 =0.73 µM, KD =0.16 µM), allowing the display of model cargos via a single mixing step. The TMV-binding peptide is specific for TMV but does not recognize free coat proteins and can therefore be used to decorate intact TMV or detect intact TMV particles in crude plant sap.

Bioconjugation Strategies for Tobacco Mild Green Mosaic Virus.

Tobacco mild green mosaic virus (TMGMV) is a plant virus closely related to Tobacco mosaic virus (TMV), sharing many of its structural and chemical features. These rod-shaped viruses, comprised of 2130 identical coat protein subunits, have been utilized as nanotechnological platforms for a myriad of applications, ranging from drug delivery to precision agriculture. This versatility for functionalization is due to their chemically active external and internal surfaces. While both viruses are similar, they do exhibit some key differences in their surface chemistry, suggesting the reactive residue distribution on TMGMV should not overlap with TMV. In this work, we focused on the establishment and refinement of chemical bioconjugation strategies to load molecules into or onto TMGMV for targeted delivery. A combination of NHS, EDC, and diazo coupling reactions in combination with click chemistry were used to modify the N-terminus, glutamic/aspartic acid residues, and tyrosines in TMGMV. We report loading with over 600 moieties per TMGMV via diazo-coupling, which is a >3-fold increase compared to previous studies. We also report that cargo can be loaded to the solvent-exposed N-terminus and carboxylates on the exterior/interior surfaces. Mass spectrometry revealed the most reactive sites to be Y12 and Y72, both tyrosine side chains are located on the exterior surface. For the carboxylates, interior E106 (66.53 %) was the most reactive for EDC-propargylamine coupled reactions, with the exterior E145 accounting for >15 % reactivity, overturning previous assumptions that only interior glutamic acid residues are accessible. A deeper understanding of the chemical properties of TMGMV further enables its functionalization and use as a multifunctional nanocarrier platform for applications in medicine and precision farming.

Multivalent Display of ApoAI Peptides on the Surface of Tobacco Mosaic Virus Nanotubes Improves Cholesterol Efflux.

Atherosclerosis is a progressive cardiovascular disease in which cholesterol-rich plaques build up within arteries, increasing the risk of thrombosis, myocardial infarction, and stroke. One promising therapeutic approach is the use of high-density lipoprotein (HDL) biomimetic formulations based on ApoAI peptides that promote cholesterol efflux from plaques, ultimately leading to cholesterol excretion. Here, we describe the multivalent display of ApoAI peptides on the surface of protein nanotubes derived from the plant virus tobacco mosaic virus (TMV) and protein nanoparticles using virus-like particles from bacteriophage Qß. Bioconjugation yielded ApoAI conjugates varying in size and morphology. We tested ABCA1-mediated cholesterol efflux using macrophage foam cells, the mitigation of reactive oxygen species in endothelial cells, and wound healing in endothelial cells. We found that the multivalent ApoAI platform, in particular the TMV-based nanotube, significantly improved the efficacy of cholesterol efflux compared to free peptides, Qß nanoparticle formulations, and traditional HDL therapy. Finally, to better understand the mechanistic basis of enhanced cholesterol efflux, we used confocal microscopy to show that while native TMV was taken up by cells, TMV-ApoAI remained at the exterior of foam cell membranes and efflux was documented using fluorescent cholesterol. Together, these data highlight that high aspect ratio materials with multivalent display of ApoAI peptides offer unique capabilities promoting efficient cholesterol efflux and may find applications in cardiovascular therapy.

A Photoacoustic Contrast Agent for miR-21 via NIR Fluorescent Hybridization Chain Reaction.

Nucleic acids are well-established biomarkers of cancer with immense value in diagnostics and basic research. However, strategies to monitor these species in tissue can be challenging due to the need for amplification of imaging signal from low analyte concentrations with high specificity. Photoacoustic (PA) imaging is gaining traction for molecular imaging of proteins, small biomolecules, and nucleic acids by coupling pulsed near-infrared (NIR) excitation with broadband acoustic detection. This work introduces a PA nucleic acid contrast agent that harnesses NIR fluorophore and quencher-tagged hybridization chain reaction (HCR) for signal amplification. This HCR probe was designed to enable contact quenching between NIR dye-quencher pairs by coercing their direct alignment when miR-21, a microRNA cancer biomarker, is detected. The probe demonstrated a ratiometric PA limit of detection of 148 pM miR-21, sequence specificity against one- and two-base mutations, and selectivity over other microRNAs. It was further tested in live human ovarian cancer (SKOV3) and noncancerous (HEK 293T) cells to exemplify in situ PA activation based on differences in endogenous miR-21 regulation (p = 0.0002). The probe was lastly tested in tissue mimicking phantoms to exemplify sustained contrast in centimeter-range depths and 85.3% photostability after 15 min of laser irradiation. The probe's miR-21-specific activation and its ability to maintain contrast in biologically relevant absorbing and scattering media support its consideration for live-cell PA microscopy and potential cancer diagnostics. Results from this probe also underscore the combined detection power between ratiometric PA signaling and strand amplification for more sensitive DNA-based PA sensors.