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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 Qβ 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 Qβ - but not CPMV - there is an improved antibody response against the target antigen using the Qβ-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 Qβ-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.

Cowpea chlorotic mottle virus (CCMV) is a positive-sense RNA virus that can be repurposed for gene delivery applications. Understanding the self-assembly process of the virus enabled to remove its genome and replace it with desired nucleic acids, and we and others have previously reported using CCMV virus-like particle (VLP) to encapsulate siRNA, mRNA, as well as CpG oligodeoxynucleotides. In this study, the CCMV VLP was applied to encapsulate two different formats of anti-miR-181a oligonucleotides: naked RNA and chemically stabilized RNA to knockdown highly regulated miR-181a in ovarian cancer cells. miR-181a expression in ovarian tumors is associated with high aggressiveness, invasiveness, resistance to chemotherapy, and overall poor prognosis. Therefore, miR-181a is an important target for ovarian cancer therapy. qPCR data and cancer cell migration assays demonstrated higher knockdown efficacy when anti-miR-181a oligonucleotides were encapsulated and delivered using the VLPs resulting in reduced cancer cell invasiveness. Importantly, delivery of anti-miR-181a oligonucleotide into cells could be achieved without the aid of a transfection agent or surface modification. These results highlight the opportunity of plant-derived VLPs as nucleic acid carriers.

Many plant virus-like particles (VLPs) utilized in nanotechnology are 30-nm icosahedrons. To expand the VLP platforms, we produced VLPs of Cytoplasmic type citrus leprosis virus (CiLV-C) in Nicotiana benthamiana. We were interested in CiLV-C because of its unique bacilliform shape (60-70 nm × 110-120 nm). The CiLV-C capsid protein (p29) gene was transferred to the pTRBO expression vector transiently expressed in leaves. Stable VLPs were formed, as confirmed by agarose gel electrophoresis, transmission electron microscopy and size exclusion chromatography. Interestingly, the morphology of the VLPs (15.8 ± 1.3 nm icosahedral particles) differed from that of the native bacilliform particles indicating that the assembly of native virions is influenced by other viral proteins and/or the packaged viral genome. The smaller CiLV-C VLPs will also be useful for structure-function studies to compare with the 30-nm icosahedrons of other VLPs.

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.

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 Qβ. 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, Qβ 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.

Ovarian cancer is the foremost cause of gynecological cancer and a major cause of cancer death in women. Treatment for advanced stage is surgical debulking followed by chemotherapy; however, most patients relapse with more aggressive and therapy-resistant tumors. There is a need to develop drug delivery approaches to deliver platinum therapies to tumors to increase efficacy while maintaining safety. Toward this goal, we utilized the protein nanotubes from the plant virus, tobacco mosaic virus (TMV), as a drug carrier. Specifically, the nanochannel of TMV was loaded with the active dication form of cisplatin (cisPtsup2+/sup), making use of the negatively charged Glu acid side chains that line the interior channel of TMV. We achieved a loading efficiency with ∼2700 cisPtsup2+/supper TMV; formulation stability was established with drug complexes stably loaded into the carrier for 2 months under refrigerated storage. TMV-cisPt maintained its efficacy against ovarian tumor cells with an ICsub50/subof ∼40 μM. TMV-cisPt exhibited superior efficacyivs/ifree cisPt in ovarian tumor mouse models using intraperitoneal ID8-Defb29/Vegf-a-Luc (mouse) tumors and subcutaneous A2780 (human) xenografts. TMV-cisPt treatment led to reduced tumor burden and increased survival. Using ID8-Defb29/Vegf-a-Luc-bearing C57BL/6 mice, we also noted reduced tumor growth when animals were treated with TMV alone, which may indicate antitumor immunity induced by the immunomodulatory nature of the plant virus nanoparticle. Biodistribution studies supported the efficacy data, showing increased cisPt accumulation within tumors when deliveredivia/ithe TMV carrierivs/ifree cisPt administration. Finally, good safety profiles were noted. The study highlights the potential of TMV as a drug carrier against cancer and points to the opportunity to explore plant viruses as chemo-immuno combination cancer therapeutics.

Ovarian cancer ranks fifth in cancer deaths amongst women, and most patients are diagnosed with late-stage and disseminated diseases. Surgical debulking and chemotherapy remove most of the tumor burden and provide a short period of remission; however, most patients experience cancer relapse and eventually succumb to the disease. Therefore, there is an urgent need for the development of vaccines to prime anti-tumor immunity and prevent its recurrence. Here we developed vaccine formulations composed of a mixture of irradiated cancer cells (ICCs, providing the antigen) and cowpea mosaic virus (CPMV) adjuvants. More specifically we compared the efficacy of co-formulated vs. mixtures of ICCs and CPMV. Specifically, we compared co-formulations where the ICCs and CPMV are bonded through natural CPMV-cell interactions or chemical coupling vs. mixtures of PEGylated CPMV and ICCs, where PEGylation of CPMV prevents ICC interactions. Flow cytometry and confocal imaging provided insights into the composition of the vaccines and their efficacy was tested using a mouse model of disseminated ovarian cancer. 67% of the mice receiving the co-formulated CPMV-ICCs survived the initial tumor challenge, and 60% of the surviving mice rejected tumors in a re-challenge experiment. In stark contrast, simple mixtures of the ICCs and (PEGylated) CPMV adjuvants were ineffective. Overall, this study highlights the importance of the co-delivery of cancer antigens and adjuvants in ovarian cancer vaccine development.

In situ vaccination for cancer immunotherapy uses intratumoral administration of small molecules, proteins, nanoparticles, or viruses that activate pathogen recognition receptors (PRRs) to reprogram the tumor microenvironment and prime systemic antitumor immunity. Cowpea mosaic virus (CPMV) is a plant virus that—while noninfectious toward mammals—activates mammalian PRRs. Application of CPMV as in situ vaccine (ISV) results in a potent and durable efficacy in tumor mouse models and canine patients; data indicate that CPMV outperforms small molecule PRR agonists and other nonrelated plant viruses and virus-like particles (VLPs). In this work, we set out to compare the potency of CPMV versus other plant viruses from the Secoviridae. We developed protocols to produce and isolate cowpea severe mosaic virus (CPSMV) and tobacco ring spot virus (TRSV) from plants. CPSMV, like CPMV, is a comovirus with genome and protein homology, while TRSV lacks homology and is from the genus nepovirus. When applied as ISV in a mouse model of dermal melanoma (using B16F10 cells and C57Bl6J mice), CPMV outperformed CPSMV and TRSV—again highlighting the unique potency of CPMV. Mechanistically, the increased potency is related to increased signaling through toll-like receptors (TLRs)—in particular, CPMV signals through TLR2, 4, and 7. Using knockout (KO) mouse models, we demonstrate here that all three plant viruses signal through the adaptor molecule MyD88—with CPSMV and TRSV predominantly activating TLR2 and 4. CPMV induced significantly more interferon β (IFNβ) compared to TRSV and CPSMV; therefore, IFNβ released upon signaling through TLR7 may be a differentiator for the observed potency of CPMV-ISV. Additionally, CPMV induced a different temporal pattern of intratumoral cytokine generation characterized by significantly increased inflammatory cytokines 4 days after the second of 2 weekly treatments, as if CPMV induced a “memory response”. This higher, longer-lasting induction of cytokines may be another key differentiator that explains the unique potency of CPMV-ISV.

Plant viral nanoparticles (plant VNPs) are promising biogenetic nanosystems for the delivery of therapeutic, immunotherapeutic, and diagnostic agents. The production of plant VNPs is simple and highly scalable through molecular farming in plants. Some of the important advances in VNP nanotechnology include genetic modification, disassembly/reassembly, and bioconjugation. Although effective, these methods often involve complex and timeconsuming multi-step protocols. Here, we report a simple and versatile supramolecular coating strategy for designing functional plant VNPs via metal–phenolic networks (MPNs). Specifically, this method gives plant viruses [e.g., tobacco mosaic virus (TMV), cowpea mosaic virus, and potato virus X] additional functionalities including photothermal transduction, photoacoustic imaging, and fluorescent labeling via different components in MPN coating [i.e., complexes of tannic acid (TA), metal ions (e.g., Fe(3+), Zr(4+), or Gd(3+)), or fluorescent dyes (e.g., rhodamine 6G and thiazole orange)]. For example, using TMV as a viral substrate by choosing Zr(4+)–TA and rhodamine 6G, fluorescence is observed peaking at 555 nm; by choosing Fe(3+)–TA coating, the photothermal conversion efficiency was increased from 0.8 to 33.2%, and the photoacoustic performance was significantly improved with a limit of detection of 17.7 μg mL(−1). We further confirmed that TMV@Fe(3+)–TA nanohybrids show good cytocompatibility and excellent cell-killing performance in photothermal therapy with 808 nm irradiation. These findings not only prove the practical benefits of this supramolecular coating for designing multifunctional and biocompatible plant VNPs but also bode well for using such materials in a variety of plant virus-based theranostic applications.

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 Qβ virus-like particles (VLPs) displaying a highly conserved epitope from the L2 protein of HPV (Qβ-HPV). Polymeric microneedle delivery of Qβ-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 Qβ VLP and its delivery technology is a plug-and-play system that could serve as a universal platform with a broad range of applications. Qβ 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.

Viral immunotherapies are being recognized in cancer treatment, with several currently approved or undergoing clinical testing. While contemporary approaches have focused on oncolytic viral therapies, our efforts center on the development of plant virus-based cancer immunotherapies. In a previous work, we demonstrated the potent efficacy of the cowpea mosaic virus (CPMV), a plant virus that does not replicate in animals, applied as an in situ vaccine. CPMV is an immunostimulatory drug candidate, and intratumoral administration remodels the tumor microenvironment leading to activation of local and systemic antitumor immunity. Efficacy has been demonstrated in multiple tumor mouse models and canine cancer patients. As wild-type CPMV is infectious toward various legumes and because shedding of infectious virus from patients may be an agricultural concern, we developed UV-inactivated CPMV (termed inCPMV) which is not infectious toward plants. We report that as a monotherapy, wild-type CPMV outperforms inCPMV in mouse models of dermal melanoma or disseminated colon cancer. Efficacy of inCPMV is less than that of CPMV and similar to that of RNA-free CPMV. Immunological investigation using knockout mice shows that inCPMV does not signal through TLR7 (toll-like receptor); structure-function studies indicate that the RNA is highly cross-linked and therefore unable to activate TLR7. Wild-type CPMV signals through TLR2, -4, and -7, whereas inCPMV more closely resembles RNA-free CPMV which signals through TLR2 and -4 only. The structural features of inCPMV explain the increased potency of wild-type CPMV through the triple pronged TLR activation. Strikingly, when inCPMV is used in combination with an anti-OX40 agonist antibody (administered systemically), exceptional efficacy was demonstrated in a bilateral B16F10 dermal melanoma model. Combination therapy, with in situ vaccination applied only into the primary tumor, controlled the progression of the secondary, untreated tumors, with 10 out of 14 animals surviving for at least 100 days post tumor challenge without development of recurrence or metastatic disease. This study highlights the potential of inCPMV as an in situ vaccine candidate and demonstrates the power of combined immunotherapy approaches. Strategic immunocombination therapies are the formula for success, and the combination of in situ vaccination strategies along with therapeutic antibodies targeting the cancer immunity cycle is a particularly powerful approach.

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

BackgroundIn situ vaccination (ISV) is a cancer immunotherapy strategy in which immunostimulatory reagents are introduced directly into a tumor to stimulate antitumor immunity both against the treated tumor and systemically against untreated tumors. Recently, we showed that cowpea mosaic virus (CPMV) is a potent multi-toll-like receptor (TLR) agonist with potent efficacy for treating tumors in mice and dogs by ISV. However, ISV with CPMV alone does not uniformly treat all mouse tumor models tested, however this can be overcome through strategic combinations. More insight is needed to delineate potency and mechanism of systemic antitumor immunity and abscopal effect.MethodWe investigated the systemic efficacy (abscopal effect) of CPMV ISV with a two-tumor mouse model using murine tumor lines B16F10, 4T1, CT26 and MC38. Flow cytometry identified changes in cell populations responsible for systemic efficacy of CPMV. Transgenic knockout mice and depleting antibodies validated the role of relevant candidate cell populations and cytokines. We evaluated these findings and engineered a multicomponent combination therapy to specifically target the candidate cell population and investigated its systemic efficacy, acquired resistance and immunological memory in mouse models.ResultsISV with CPMV induces systemic antitumor T-cell-mediated immunity that inhibits growth of untreated tumors and requires conventional type-1 dendritic cells (cDC1s). Furthermore, using multiple tumor mouse models resistant to anti-programmed death 1 (PD-1) therapy, we tested the hypothesis that CPMV along with local activation of antigen-presenting cells with agonistic anti-CD40 can synergize and strengthen antitumor efficacy. Indeed, this combination ISV strategy induces an influx of CD8+T cells, triggers regression in both treated local and untreated distant tumors and potentiates tumor responses to anti-PD-1 therapy. Moreover, serial ISV overcomes resistance to anti-PD-1 therapy and establishes tumor-specific immunological memory.ConclusionsThese findings provide new insights into in situ TLR activation and cDC1 recruitment as effective strategies to overcome resistance to immunotherapy in treated and untreated tumors.

Filamentous nanomaterials are flexible with a high aspect ratio, conferring unique mechanical, electromagnetic, and optical properties; promoting tissue penetration; and allowing the formation of hierarchical superstructures. The fabrication of synthetic nanofilaments with uniform properties is challenging, but this can be addressed by the use of filamentous plant viruses such as potato virus X (PVX), which are produced as monodisperse structures from a genetic template. To take advantage of PVX without risks to agriculture and the environment, it is necessary to inactivate the virus efficiently without disrupting its chemical and material properties. Herein, we report experiments showing that PVX can be completely inactivated by exposure to UV irradiation (0.5 J cm

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 Qβ. 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 Qβ VLPs could be stockpiled, and epitopes exchanged to target new mutants or emergent diseases as the need arises.

Cancer is an unprecedented proliferation of cells leading to abnormalities in differentiation and maturation. Treatment of primary and metastatic cancer is challenging. In addition to surgery, chemotherapy and radiation therapies have been conventionally used; however, they suffer from severe toxicity and non-specificity. Immunotherapy, the science of programming the body's own defense system against cancer has gained tremendous attention in the last few decades. However, partial immunogenic stimulation, premature degradation and inability to activate dendritic and helper T cells has resulted in limited clinical success. The era of nanomedicine has brought about several breakthroughs in various pharmaceutical and biomedical fields. Hereby, we review and discuss the interplay of tumor microenvironment (TME) and the immunological cascade and how they can be employed to develop nanoparticle-based cancer vaccines and immunotherapies. Nanoparticles composed of lipids, polymers and inorganic materials contain useful properties suitable for vaccine development. Proteinaceous vaccines derived from mammalian viruses, bacteriophages and plant viruses also have unique advantages due to their immunomodulation capabilities. This review accounts for all such considerations. Additionally, we explore how attributes of nanotechnology can be utilized to develop successful nanomedicine-based vaccines for cancer therapy. This article is categorized under: Nanotechnology Approaches to BiologyNanoscale Systems in Biology Therapeutic Approaches and Drug DiscoveryNanomedicine for Oncologic Disease.

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 Qβ (Qβ-PPCs) as PEG alternatives. Lyz-PPCs retain nearly 100% bioactivity and Qβ-PPCs exhibit up to 35% decrease in protein immunogenicity. Qβ-PPCs derived from NB-PEG show no reduction in recognition by anti-PEG antibodies while Qβ-PPCs derived from NB-Zwit show >95% reduction as compared with Qβ-PEG. This work demonstrates a new method for PPC synthesis and the utility of grafting from PPCs to evade immune recognition.

Antimicrobial resistance is a global health threat that is exacerbated by the overuse and misuse of antibiotics in medicine and agriculture. As an alternative to conventional antimicrobial drugs, phage therapy involves the treatment of infected patients with a bacteriophage that naturally destroys bacterial pathogens. With the re-emergence of phage therapy, novel tools are needed to study phages. In this work we set out to screen and isolate peptide candidates that bind to phages and act as affinity tags. Such peptides functionalized with an imaging agent could serves as versatile tools for tracking and imaging of phages. Specifically, we screened a phage display library for peptides that bind to the Good Vibes phage (GV), which lyses the bacterial pathogen Pseudomonas aeruginosa. Isolated monoclonal library phages featured a highly conserved consensus motif, LPPIXRX. The corresponding peptide WDLPPIGRLSGN was synthesized with a GGGSK linker and conjugated to cyanine 5 or biotin. The specific binding of the LPPIXRX motif to GV in vitro was confirmed using an enzyme-linked immunosorbent assay. We demonstrated imaging and tracking of GV in bacterial populations using the fluorescent targeting peptide and flow cytometry. In conclusion, we developed fluorescent labeled peptides that can bind to bacteriophage GV specifically, which may enable real-time analysis of phage in vivo and monitor the efficacy of phage therapy.

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.

The plant virus cowpea mosaic virus (CPMV) is a natural nanocarrier that has been developed as a platform technology for the delivery of various payloads including peptide epitopes for vaccines, contrast agents for imaging, and drugs for therapy. Genetic fusion and chemical conjugations are the mainstay approaches to load the active ingredient to the exterior and/or interior of CPMV. However, these methods have limitations; genetic engineering is limited to biologics, and chemical alteration often requires multistep reactions with modification of both CPMV and the active ingredient. Either method can also result in particle instability. Therefore, to provide an alternate path toward CPMV functionalization, we report the isolation of peptides that specifically bind to CPMV, termed CPMV-binding peptides (CBP). We used a commercial M13 phage display 7-mer peptide library to pan for and select peptides that selectively bind to CPMV. Biopanning and characterization of lead candidates resulted in isolation of the motif "GWRVSEF/L" as the CPMV-specific motif with phenylalanine (F) at the seventh position being stronger than leucine (L). Specificity to CPMV was demonstrated, and cross-reactivity toward other plant viruses was not observed. To demonstrate cargo loading, GWRVSEF was tagged with biotin, fluorescein isothiocyanate (FITC), and a human epidermal growth factor receptor 2 (HER2)-specific targeting peptide ligand. Display of the active ingredient was confirmed, and utility of tagged and targeted CPMV in cell binding assays was demonstrated. The CBP functionalization strategy offers a new avenue for CPMV nanoparticle functionalization and should offer a versatile tool to add active ingredients that otherwise may be difficult to conjugate or display.

Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a particular coronavirus strain responsible for the coronavirus disease 2019 (COVID-19), accounting for more than 3.1 million deaths worldwide. Several health-related strategies have been successfully developed to contain the rapidly-spreading virus across the globe, toward reduction of both disease burden and infection rates. Particularly, attention has been focused on either the development of novel drugs and vaccines, or by adapting already-existing drugs for COVID-19 treatment, mobilizing huge efforts to block disease progression and to overcome the shortage of effective measures available at this point. Areas covered: This perspective covers the breakthrough of multifunctional biomimetic cell membrane-based nanoparticles as next-generation nanosystems for cutting-edge COVID-19 therapeutics and vaccination, specifically cell membrane-derived nanovesicles and cell membrane-coated nanoparticles, both tailorable cell membrane-based nanosystems enriched with the surface repertoire of native cell membranes, toward maximized biointerfacing, immune evasion, cell targeting and cell-mimicking properties. Expert opinion: Nano-based approaches have received widespread interest regarding enhanced antigen delivery, prolonged blood circulation half-life and controlled release of drugs. Cell membrane-based nanoparticles comprise interesting antiviral multifunctional nanoplatforms for blocking SARS-CoV-2 binding to host cells, reducing inflammation through cytokine neutralization and improving drug delivery toward COVID-19 treatment.

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., 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.

Antimicrobial resistance is a global health threat that is exacerbated by the overuse and misuse of antibiotics in medicine and agriculture. As an alternative to conventional antimicrobial drugs, phage therapy involves the treatment of infected patients with a bacteriophage that naturally destroys bacterial pathogens. With the re-emergence of phage therapy, novel tools are needed to study phages. In this work we set out to screen and isolate peptide candidates that bind to phages and act as affinity tags. Such peptides functionalized with an imaging agent could serves as versatile tools for tracking and imaging of phages. Specifically, we screened a phage display library for peptides that bind to the Good Vibes phage (GV), which lyses the bacterial pathogen

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 a3-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 for15 % 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.

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.

Engineered living materials (ELMs) that incorporate living organisms and synthetic materials enable advanced functional properties. Here, we seek to create plant cyborgs by combining plants or plant tissues with stimuli-responsive polymeric materials. Plant tissues with integrated shape control may find applications in regenerative medicine, and the shape control of living plants enables another dimension of adaptability and response to environmental threats, which can be applied to next-generation precision farming. In this work, we develop chemistry to integrate stimuli-responsive poly(

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.

Nanoparticle (NP)-based drug delivery systems are promising in anti-cancer therapy, capable of delivering cargo with superior selectivity and achieving enhanced tumor accumulation compared to small-molecule therapeutics. As more efforts are being devoted to NP development, molecular polymer bottlebrushes (MPBs) have gained attention as a potential drug delivery vehicle. To date, the influence of various MPB parameters such as size, shape, and surface charge in determining tumor penetrability have been systematically probed. However, the role of amphiphilicity, specifically the hydrophilic-hydrophobic balance, remains unexplored. In this study, a series of MPBs are employed with varied hydrophobicity levels to reveal a dependence between MPB composition, cell association and tumor homing. The data indicates that increasing levels of hydrophobicity in MPBs (to a certain level) demonstrate only marginal effects in vitro but reveals enhanced tumor homing in a mouse model of ovarian cancer in vivo, where more hydrophilic MPBs exhibit low tissue deposition and low tumor homing. In contrast, more hydrophobic MPBs show significant tumor accumulation and homing due to their engineered hydrophobicity.