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.

Photothermal immunotherapy of melanoma using TLR-7 agonist laden tobacco mosaic virus with polydopamine coat.

Photothermal therapy (PTT) is a promising cancer treatment that debulks tumors locally while priming immune responses. However, PTT as a standalone treatment approach often has limited systemic efficacy, motivating the development of synergistic combination approaches. Toward this goal, herein, the tobacco mosaic virus (TMV) was loaded with a small molecule immunomodulator, toll-like receptor 7 agonist (1V209), and its surface was coated with photothermal biopolymer polydopamine (PDA). The resulting 1V209-laden and PDA-coated TMV was used to treat B16F10 dermal melanoma in C57BL/6 mice. 1V209-TMV-PDA was intratumorally injected and irradiated using an 808-nm near infrared laser. 60 % of the mice receiving PTT with intratumoral 1V209-TMV-PDA + laser remained alive at the end point - in contrast to only 20 % survivors were observed in the control group. Immunological analysis indicates systemic anti-tumor immunity being induced by the combination therapy with a greater number of tumor-specific T cells (as determined by a splenocyte assay). This study highlights the potential of TMV versatility as a multifunctional nano-platform for combined PTT-immunotherapy.

Biological and evolutionary concepts for nanoscale engineering: Viruses as natural nanoparticles have great potential for a wide range of nanoscale products.

Biological materials are a rich resource for nanoscale engineering. Their structure is easily accessible via their DNA and they were optimised through evolution to fulfill their function.

Bioinspired Shielding Strategies for Nanoparticle Drug Delivery Applications.

Nanoparticle delivery systems offer advantages over free drugs, in that they increase solubility and biocompatibility. Nanoparticles can deliver a high payload of therapeutic molecules while limiting off-target side effects. Therefore, delivery of an existing drug with a nanoparticle frequently results in an increased therapeutic index. Whether of synthetic or biologic origin, nanoparticle surface coatings are often required to reduce immune clearance and thereby increase circulation times allowing the carriers to reach their target site. To this end, polyethylene glycol (PEG) has long been used, with several PEGylated products reaching clinical use. Unfortunately, the growing use of PEG in consumer products has led to an increasing prevalence of PEG-specific antibodies in the human population, which in turn has fueled the search for alternative coating strategies. This review highlights alternative bioinspired nanoparticle shielding strategies, which may be more beneficial moving forward than PEG and other synthetic polymer coatings.

Polymer Structure and Conformation Alter the Antigenicity of Virus-like Particle-Polymer Conjugates.

Covalent conjugation of water-soluble polymers to proteins is critical for evading immune surveillance in the field of biopharmaceuticals. The most common and long-standing polymer modification is the attachment of methoxypoly(ethylene glycol) (mPEG), termed PEGylation, which has led to several clinically approved pharmaceuticals. Recent data indicate that brush-type polymers significantly enhance in vitro and in vivo properties. Herein, the polymer conformation of poly(ethylene glycol) is detailed and compared with those of water-soluble polyacrylate and polynorbornene (PNB) when attached to icosahedral virus-like particles. Small-angle neutron scattering reveals vastly different polymer conformations of the multivalent conjugates. Immune recognition of conjugated particles was evaluated versus PEGylated particles, and PNB conjugation demonstrated the most effective shielding from antibody recognition.

Optical and Magnetic Resonance Imaging Using Fluorous Colloidal Nanoparticles.

Improved imaging of cancerous tissue has the potential to aid prognosis and improve patient outcome through longitudinal imaging of treatment response and disease progression. While nuclear imaging has made headway in cancer imaging, fluorinated tracers that enable magnetic resonance imaging (19F MRI) hold promise, particularly for repeated imaging sessions because nonionizing radiation is used. Fluorine MRI detects molecular signatures by imaging a fluorinated tracer and takes advantage of the spatial and anatomical resolution afforded by MRI. This manuscript describes a fluorous polymeric nanoparticle that is capable of 19F MR imaging and fluorescent tracking for in vitro and in vivo monitoring of immune cells and cancerous tissue. The fluorous particle is derived from low-molecular-weight amphiphilic copolymers that self-assemble into micelles with a hydrodynamic diameter of 260 nm. The polymer is MR-active at concentrations as low as 2.1 mM in phantom imaging studies. The fluorinated particle demonstrated rapid uptake into immune cells for potential cell-tracking or delineation of the tumor microenvironment and showed negligible toxicity. Systemic administration indicates significant uptake into two tumor types, triple-negative breast cancer and ovarian cancer, with little accumulation in off-target tissue. These results indicate a robust platform imaging agent capable of immune cell tracking and systemic disease monitoring with exceptional uptake of the nanoparticle in multiple cancer models.

The Protein Corona of Plant Virus Nanoparticles Influences their Dispersion Properties, Cellular Interactions, and In Vivo Fates.

Biomolecules in bodily fluids such as plasma can adsorb to the surface of nanoparticles and influence their biological properties. This phenomenon, known as the protein corona, is well established in the field of synthetic nanotechnology but has not been described in the context of plant virus nanoparticles (VNPs). The interaction between VNPs derived from Tobacco mosaic virus (TMV) and plasma proteins is investigated, and it is found that the VNP protein corona is significantly less abundant compared to the corona of synthetic particles. The formed corona is dominated by complement proteins and immunoglobulins, the binding of which can be reduced by PEGylating the VNP surface. The impact of the VNP protein corona on molecular recognition and cell targeting in the context of cancer and thrombosis is investigated. A library of functionalized TMV rods with polyethylene glycol (PEG) and peptide ligands targeting integrins or fibrin(ogen) show different dispersion properties, cellular interactions, and in vivo fates depending on the properties of the protein corona, influencing target specificity, and non-specific scavenging by macrophages. Our results provide insight into the in vivo properties of VNPs and suggest that the protein corona effect should be considered during the development of efficacious, targeted VNP formulations.

Free-Standing, Nanopatterned Janus Membranes of Conducting Polymer-Virus Nanoparticle Arrays.

Nanostructured mesoscale materials find wide-ranging applications in medicine and energy. Top-down manufacturing schemes are limited by the smallest dimension accessible; therefore, we set out to study a bottom-up approach mimicking biological systems, which self-assemble into systems that orchestrate complex energy conversion functionalities. Inspired by nature, we turned toward protein-based nanoparticle structures formed by plant viruses, specifically the cowpea mosaic virus (CPMV). We report the formation of hierarchical CPMV nanoparticle assemblies on colloidal-patterned, conducting polymer arrays using a protocol combining colloidal lithography, electrochemical polymerization, and electrostatic adsorption. In this approach, a hexagonally close-packed array of polystyrene microspheres was assembled on a conductive electrode to function as the sacrificial colloidal template. A thin layer of conducting polypyrrole material was electrodeposited within the interstices of the colloidal microspheres and monitored in situ using electrochemical quartz crystal microbalance with dissipation (EC-QCM-D). Etching the template revealed an inverse opaline conducting polymer pattern capable of forming strong electrostatic interactions with CPMV and therefore enabling immobilization of CPMV on the surface. The CPMV-polymer films were characterized by atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). Furthermore, molecular probe diffusion experiments revealed selective ion transport properties as a function of the presence of the CPMV nanoparticles on the surface. Lastly, by utilizing its electromechanical behavior, the polymer/protein membrane was electrochemically released as a free-standing film, which can potentially be used for developing high surface area cargo delivery systems, stimuli-responsive plasmonic devices, and chemical and biological sensors.

Fluorescent nanodiamonds embedded in biocompatible translucent shells.

High pressure high temperature (HPHT) nanodiamonds (NDs) represent extremely promising materials for construction of fluorescent nanoprobes and nanosensors. However, some properties of bare NDs limit their direct use in these applications: they precipitate in biological solutions, only a limited set of bio-orthogonal conjugation techniques is available and the accessible material is greatly polydisperse in shape. In this work, we encapsulate bright 30-nm fluorescent nanodiamonds (FNDs) in 10-20-nm thick translucent (i.e., not altering FND fluorescence) silica shells, yielding monodisperse near-spherical particles of mean diameter 66 nm. High yield modification of the shells with PEG chains stabilizes the particles in ionic solutions, making them applicable in biological environments. We further modify the opposite ends of PEG chains with fluorescent dyes or vectoring peptide using click chemistry. High conversion of this bio-orthogonal coupling yielded circa 2000 dye or peptide molecules on a single FND. We demonstrate the superior properties of these particles by in vitro interaction with human prostate cancer cells: while bare nanodiamonds strongly aggregate in the buffer and adsorb onto the cell membrane, the shell encapsulated NDs do not adsorb nonspecifically and they penetrate inside the cells.

Poly(Oxanorbornene)-Protein Conjugates Prepared by Grafting-to ROMP as Alternatives for PEG.

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.

Study of uricase-polynorbornene conjugates derived from grafting-from ring-opening metathesis polymerization.

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.

Grafting-from Synthesis of Plant-Polynorbornene Biohybrid Materials.

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.

Peptide-Driven Proton Sponge Nano-Assembly for Imaging and Triggering Lysosome-Regulated Immunogenic Cancer Cell Death.

Triggering lysosome-regulated immunogenic cell death (ICD, e.g., pyroptosis and necroptosis) with nanomedicines is an emerging approach for turning an "immune-cold" tumor "hot"-a key challenge faced by cancer immunotherapies. Proton sponge such as high-molecular-weight branched polyethylenimine (PEI) is excellent at rupturing lysosomes, but its therapeutic application is hindered by uncontrollable toxicity due to fixed charge density and poor understanding of resulted cell death mechanism. Here, a series of proton sponge nano-assemblies (PSNAs) with self-assembly controllable surface charge density and cell cytotoxicity are created. Such PSNAs are constructed via low-molecular-weight branched PEI covalently bound to self-assembling peptides carrying tetraphenylethene pyridinium (PyTPE, an aggregation-induced emission-based luminogen). Assembly of PEI assisted by the self-assembling peptide-PyTPE leads to enhanced surface positive charges and cell cytotoxicity of PSNA. The self-assembly tendency of PSNAs is further optimized by tuning hydrophilic and hydrophobic components within the peptide, thus resulting in the PSNA with the highest fluorescence, positive surface charge density, cell uptake, and cancer cell cytotoxicity. Systematic cell death mechanistic studies reveal that the lysosome rupturing-regulated pyroptosis and necroptosis are at least two causes of cell death. Tumor cells undergoing PSNA-triggered ICD activate immune cells, suggesting the great potential of PSNAs to trigger anticancer immunity.

Increased tumor homing and tissue penetration of the filamentous plant viral nanoparticle Potato virus X.

Nanomaterials with elongated architectures have been shown to possess differential tumor homing properties compared to their spherical counterparts. Here, we investigate whether this phenomenon is mirrored by plant viral nanoparticles that are filamentous (Potato virus X) or spherical (Cowpea mosaic virus). Our studies demonstrate that Potato virus X (PVX) and Cowpea mosaic virus (CPMV) show distinct biodistribution profiles and differ in their tumor homing and penetration efficiency. Analogous to what is seen with inorganic nanomaterials, PVX shows enhanced tumor homing and tissue penetration. Human tumor xenografts exhibit higher uptake of PEGylated filamentous PVX compared to CPMV, particularly in the core of the tumor. This is supported by immunohistochemical analysis of the tumor sections, which indicates greater penetration and accumulation of PVX within the tumor tissues. The enhanced tumor homing and retention properties of PVX along with its higher payload carrying capacity make it a potentially superior platform for applications in cancer drug delivery and imaging applications.

A co-formulated vaccine of irradiated cancer cells and cowpea mosaic virus improves ovarian cancer rejection.

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.

Molecularly Stimuli-Responsive Self-Assembled Peptide Nanoparticles for Targeted Imaging and Therapy.

Self-assembly has emerged as an extensively used method for constructing biomaterials with sizes ranging from nanometers to micrometers. Peptides have been extensively investigated for self-assembly. They are widely applied owing to their desirable biocompatibility, biodegradability, and tunable architecture. The development of peptide-based nanoparticles often requires complex synthetic processes involving chemical modification and supramolecular self-assembly. Stimuli-responsive peptide nanoparticles, also termed "smart" nanoparticles, capable of conformational and chemical changes in response to stimuli, have emerged as a class of promising materials. These smart nanoparticles find a diverse range of biomedical applications, including drug delivery, diagnostics, and biosensors. Stimuli-responsive systems include external stimuli (such as light, temperature, ultrasound, and magnetic fields) and internal stimuli (such as pH, redox environment, salt concentration, and biomarkers), facilitating the generation of a library of self-assembled biomaterials for biomedical imaging and therapy. Thus, in this review, we mainly focus on peptide-based nanoparticles built by self-assembly strategy and systematically discuss their mechanisms in response to various stimuli. Furthermore, we summarize the diverse range of biomedical applications of peptide-based nanomaterials, including diagnosis and therapy, to demonstrate their potential for medical translation.

Interior engineering of a viral nanoparticle and its tumor homing properties.

The development of multifunctional nanoparticles for medical applications is of growing technological interest. A single formulation containing imaging and/or drug moieties that is also capable of preferential uptake in specific cells would greatly enhance diagnostics and treatments. There is growing interest in plant-derived viral nanoparticles (VNPs) and establishing new platform technologies based on these nanoparticles inspired by nature. Cowpea mosaic virus (CPMV) serves as the standard model for VNPs. Although exterior surface modification is well-known and has been comprehensively studied, little is known of interior modification. Additional functionality conferred by the capability for interior engineering would be of great benefit toward the ultimate goal of targeted drug delivery. Here, we examined the capacity of empty CPMV (eCPMV) particles devoid of RNA to encapsulate a wide variety of molecules. We systematically investigated the conjugation of fluorophores, biotin affinity tags, large molecular weight polymers such as poly(ethylene glycol) (PEG), and various peptides through targeting reactive cysteines displayed selectively on the interior surface. Several methods are described that mutually confirm specific functionalization of the interior. Finally, CPMV and eCPMV were labeled with near-infrared fluorophores and studied side-by-side in vitro and in vivo. Passive tumor targeting via the enhanced permeability and retention effect and optical imaging were confirmed using a preclinical mouse model of colon cancer. The results of our studies lay the foundation for the development of the eCPMV platform in a range of biomedical applications.

Tuning the Hydrophilic-Hydrophobic Balance of Molecular Polymer Bottlebrushes Enhances their Tumor Homing Properties.

Nanoparticle (NP)-based drug delivery systems are promising in anticancer 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.

Intravital imaging of human prostate cancer using viral nanoparticles targeted to gastrin-releasing Peptide receptors.

Multivalent nanoparticles have several key advantages in terms of solubility, binding avidity, and uptake, making them particularly well suited to molecular imaging applications. Herein is reported the stepwise synthesis and characterization of NIR viral nanoparticles targeted to gastrin-releasing peptide receptors that are over-expressed in human prostate cancers. The pan-bombesin analogue, [ß-Ala11, Phe13, Nle14]bombesin-(7-14), is conjugated to cowpea mosaic virus particles functionalized with an NIR dye (Alexa Fluor 647) and polyethylene glycol (PEG) using the copper(I)-catalyzed azide-alkyne cycloaddition reaction. Targeting and uptake in human PC-3 prostate cells is demonstrated in vitro. Tumor homing is observed using human prostate tumor xenografts on the chicken chorioallantoic membrane model using intravital imaging. Further development of this viral nanoparticle platform may open the door to potential clinical noninvasive molecular imaging strategies.

Potato virus X as a novel platform for potential biomedical applications.

We demonstrate that nanoparticles formed from the rod-shaped plant virus Potato virus X (PVX) can serve as a novel platform for biomedical applications. Bioconjugation protocols including amine modification and "click" chemistry allowed the efficient functionalization of PVX with biotins, dyes, and PEGs. Fluorescent-labeled and PEGylated PVX particles revealed that different fluorescent labels have a profound effect on PVX-cell interactions. Applying bioconjugation chemistries to PVX opens the door for chemical functionalization with targeting and therapeutic molecules.