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.

Dissolving Microneedle Delivery of a Prophylactic HPV Vaccine.

Prophylactic vaccines capable of preventing human papillomavirus (HPV) infections are still inaccessible to a vast majority of the global population due to their high cost and challenges related to multiple administrations performed in a medical setting. In an effort to improve distribution and administration, we have developed dissolvable microneedles loaded with a thermally stable HPV vaccine candidate consisting of Qß virus-like particles (VLPs) displaying a highly conserved epitope from the L2 protein of HPV (Qß-HPV). Polymeric microneedle delivery of Qß-HPV produces similar amounts of anti-HPV16 L2 IgG antibodies compared to traditional subcutaneous injection while delivering a much smaller amount of intradermal dose. However, a dose sparing effect was found. Furthermore, immunization yielded neutralizing antibody responses in a HPV pseudovirus assay. The vaccine candidate was confirmed to be stable at room temperature after storage for several months, potentially mitigating many of the challenges associated with cold-chain distribution. The ease of self-administration and minimal invasiveness of such microneedle patch vaccines may enable wide-scale distribution of the HPV vaccine and lead to higher patient compliance. The Qß VLP and its delivery technology is a plug-and-play system that could serve as a universal platform with a broad range of applications. Qß VLPs may be stockpiled for conjugation to a wide range of epitopes, which are then packaged and delivered directly to the patient via noninvasive microneedle patches. Such a system paves the way for rapid distribution and self-administration of vaccines.

Trivalent Subunit Vaccine Candidates for COVID-19 and Their Delivery Devices.

The COVID-19 pandemic highlights the need for platform technologies enabling rapid development of vaccines for emerging viral diseases. The current vaccines target the SARS-CoV-2 spike (S) protein and thus far have shown tremendous efficacy. However, the need for cold-chain distribution, a prime-boost administration schedule, and the emergence of variants of concern (VOCs) call for diligence in novel SARS-CoV-2 vaccine approaches. We studied 13 peptide epitopes from SARS-CoV-2 and identified three neutralizing epitopes that are highly conserved among the VOCs. Monovalent and trivalent COVID-19 vaccine candidates were formulated by chemical conjugation of the peptide epitopes to cowpea mosaic virus (CPMV) nanoparticles and virus-like particles (VLPs) derived from bacteriophage Qß. Efficacy of this approach was validated first using soluble vaccine candidates as solo or trivalent mixtures and subcutaneous prime-boost injection. The high thermal stability of our vaccine candidates allowed for formulation into single-dose injectable slow-release polymer implants, manufactured by melt extrusion, as well as microneedle (MN) patches, obtained through casting into micromolds, for prime-boost self-administration. Immunization of mice yielded high titers of antibodies against the target epitope and S protein, and data confirms that antibodies block receptor binding and neutralize SARS-CoV and SARS-CoV-2 against infection of human cells. We present a nanotechnology vaccine platform that is stable outside the cold-chain and can be formulated into delivery devices enabling single administration or self-administration. CPMV or Qß VLPs could be stockpiled, and epitopes exchanged to target new mutants or emergent diseases as the need arises.

Polymer Chemistry for Haptics, Soft Robotics, and Human-Machine Interfaces.

Progress in the field of soft devices-i.e., haptics, robotics, and human-machine interfaces (HRHMIs)-has its basis in the science of polymeric materials and chemical synthesis. However, in examining the relevant literature, we find that most developments have been enabled by off-the-shelf materials used either alone or as components of physical blends and composites. In this Progress Report, we take the position that a greater awareness of the capabilities of synthetic chemistry will accelerate the capabilities of HRHMIs. Conversely, an awareness of the applications sought by engineers working in this area may spark the development of new molecular designs and synthetic methodologies by chemists. We highlight several applications of active, stimuli-responsive polymers, which have demonstrated or shown potential use in HRHMIs. These materials share the fact that they are products of state-of-the-art synthetic techniques. The Progress Report is thus organized by the chemistry by which the materials were synthesized, including controlled radical polymerization, metal-mediated cross-coupling polymerization, ring-opening polymerization, various strategies for crosslinking, and hybrid approaches. These methods can afford polymers with multiple properties (i.e. conductivity, stimuli-responsiveness, self-healing and degradable abilities, biocompatibility, adhesiveness, and mechanical robustness) that are of great interest to scientists and engineers concerned with soft devices for human interaction.

Freeze-Drying To Produce Efficacious CPMV Virus-like Particles.

In situ cancer vaccination that uses immune stimulating agents is revolutionizing the way that cancer is treated. In this realm, viruses and noninfectious virus-like particles have gained significant traction in reprogramming the immune system to recognize and eliminate malignancies. Recently, cowpea mosaic virus-like particles (VLPs) have shown exceptional promise in their ability to fight a variety of cancers. However, the current methods used to produce CPMV VLPs rely on agroinfiltration in plants. These protocols remain complicated and labor intensive and have the potential to introduce unwanted immunostimulatory agents, like lipopolysaccharides. This Letter describes a simple "post-processing" method to remove RNA from wild-type CPMV, while retaining the structure and function of the capsid. Lyophilization was able to eject encapsulated RNA to form lyo-eCPMV and, when purified, eliminated nearly all traces of encapsulated RNA. Lyo-eCPMV was characterized by cryo-electron microscopy single particle reconstruction to confirm the structural integrity of the viral capsid. Finally, lyo-eCPMV showed  equivalent anticancer efficacy as eCPMV, produced by agroinfiltration, when using an invasive melanoma model. These results describe a straightforward method to prepare CPMV VLPs from infectious virions.

Cell Engineering with Functional Poly(oxanorbornene) Block Copolymers.

Cell-based therapies are gaining prominence in treating a wide variety of diseases and using synthetic polymers to manipulate these cells provides an opportunity to impart function that could not be achieved using solely genetic means. Herein, we describe the utility of functional block copolymers synthesized by ring-opening metathesis polymerization (ROMP) that can insert directly into the cell membrane via the incorporation of long alkyl chains into a short polymer block leading to non-covalent, hydrophobic interactions with the lipid bilayer. Furthermore, we demonstrate that these polymers can be imbued with advanced functionalities. A photosensitizer was incorporated into these polymers to enable spatially controlled cell death by the localized generation of 1 O2 at the cell surface in response to red-light irradiation. In a broader context, we believe our polymer insertion strategy could be used as a general methodology to impart functionality onto cell-surfaces.

Polymeric Interventions for Microbial Infections: A Review.

The world is facing a growing crisis of microbial infections, where resistant strains are rapidly outpacing the development of new therapeutics. In an effort to combat this, the polymer community is developing new ways to improve upon drug delivery, synthesizing novel antimicrobial polymers, and using polymer technology to harness combination therapies. This review focuses primarily on the use of polymers to treat both bacterial and fungal infections in recent years. A bevy of work has illustrated that polymer technologies can have a huge impact in treating bacterial infections. However, harnessing polymers to deliver antifungals or as stand-alone therapeutics lags far behind that of interventions for bacterial infections. Fungal infections can be crippling to both human health and the agricultural community, making this area ripe for drug delivery technologies. This review describes recent work and highlights opportunities for bacterial and fungal treatment using soft matter.

pH Responsive Doxorubicin Delivery by Fluorous Polymers for Cancer Treatment.

Polymeric nanoparticles have emerged as valuable drug delivery vehicles as they improve solubility of hydrophobic drugs, enhance circulation lifetime, and can improve the biodistribution profile of small-molecule therapeutics. These nanoparticles can take on a host of polymer architectures including polymersomes, hyperbranched nanoparticles, and dendrimers. We have recently reported that simple low molecular weight fluorous copolymers can self-assemble into nanoparticles and show exceptional passive targeting into multiple tumor models. Given the favorable biodistribution of these particles, we sought to develop systems that enable selective delivery in acidic environments, such as the tumor microenvironment or the lysosomal compartment. In this report, we describe the synthesis and in vitro biological studies of a pH-responsive doxorubicin (DOX) fluorous polymer conjugate. A propargyl DOX hydrazone was synthesized and covalently attached to a water-dispersible fluorous polymer composed of trifluoroethyl methacrylate (TFEMA) and oligo(ethylene glycol) methyl ether methacrylate (OEGMEMA) using the ligand-accelerated copper-catalyzed azide-alkyne cycloaddition. Driven by the high fluorine content of the copolymer carrier, the DOX-copolymer formed stable micelles under aqueous conditions with a hydrodynamic diameter of 250 nm. The DOX-copolymer showed internalization into multiple in vitro models for breast and ovarian cancer. Cytotoxicity assays demonstrated efficacy in both breast and ovarian cancer with overall efficacy being highly dependent on the cell line chosen. Taken together, these results present a platform for the pH-triggered delivery of DOX from a fluorous micelle carrier effective against multiple cancer models in vitro.

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.

Quantitative Molecular Imaging with a Single Gd-Based Contrast Agent Reveals Specific Tumor Binding and Retention in Vivo.

Magnetic resonance imaging (MRI) has become an indispensable tool in the diagnosis and treatment of many diseases, especially cancer. However, the poor sensitivity of MRI relative to other imaging modalities, such as PET, has hindered the development and clinical use of molecular MRI contrast agents that could provide vital diagnostic information by specifically locating a molecular target altered in the disease process. This work describes the specific and sustained in vivo binding and retention of a protein tyrosine phosphatase mu (PTPµ)-targeted, molecular magnetic resonance (MR) contrast agent with a single gadolinium (Gd) chelate using a quantitative MRI T1 mapping technique in glioma xenografts. Quantitative T1 mapping is an imaging method used to measure the longitudinal relaxation time, the T1 relaxation time, of protons in a magnetic field after excitation by a radiofrequency pulse. T1 relaxation times can in turn be used to calculate the concentration of a gadolinium-containing contrast agent in a region of interest, thereby allowing the retention or clearance of an agent to be quantified. In this context, retention is a measure of molecular contrast agent binding. Using conventional peptide chemistry, a PTPµ-targeted peptide was linked to a chelator that had been conjugated to a lysine residue. Following complexation with Gd, this PTPµ-targeted molecular contrast agent containing a single Gd ion showed significant tumor enhancement and a sustained increase in Gd concentration in both heterotopic and orthotopic tumors using dynamic quantitative MRI. This single Gd-containing PTPµ agent was more effective than our previous version with three Gd ions. Differences between nonspecific and specific agents, due to specific tumor binding, can be determined within the first 30 min after agent administration by examining clearance rates. This more facile chemistry, when combined with quantitative MR techniques, allows for widespread adoption by academic and commercial entities in the field of molecular MRI ultimately leading to improved detection of disease.

PEGylated Dendrimers as Drug Delivery Vehicles for the Photosensitizer Silicon Phthalocyanine Pc 4 for Candidal Infections.

Fungi account for billions of infections worldwide. The second most prominent causative agent for fungal infections is Candida albicans (C. albicans). As strains of fungi become resistant to antifungal medications, new treatment modalities must be investigated to combat these infections. One approach is to employ photodynamic therapy (PDT). PDT utilizes a photosensitizer, light, and cellular O2 to produce reactive oxygen species (ROS), which then induce oxidative stress resulting in apoptosis. Silicon phthalocyanine Pc 4 is a photosensitizer that has exhibited success in clinical trials for a myriad of skin diseases. The hydrophobic nature of Pc 4, however, poses significant formulation and delivery challenges in the use of this therapy. To mitigate these concerns, a drug delivery vehicle was synthesized to better formulate Pc 4 into a viable PDT agent for treating fungal infections. Utilizing poly(amidoamine) dendrimers as the framework for the vehicle, ∼13% of the amine chain ends were PEGylated to promote water solubility and deter nonspecific adsorption. In vitro studies with C. albicans demonstrate that the potency of Pc 4 was not hindered by the dendrimer vehicle. Encapsulated Pc 4 was able to effectively generate ROS and obliterate fungal pathogens upon photoactivation. The results presented within describe a nanoparticulate delivery vehicle for Pc 4 that readily kills drug-resistant C. albicans and eliminates solvent toxicity, thus, improving formulation characteristics for the hydrophobic photosensitizer.

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.

"Graft-to" Protein/Polymer Conjugates Using Polynorbornene Block Copolymers.

A series of water-soluble polynorbornene block copolymers prepared via Ring-Opening Metathesis Polymerization (ROMP) were grafted to proteins to form ROMP-derived bioconjugates. ROMP afforded low-dispersity polymers and allowed for strict control over polymer molecular weight and architecture. The polymers consisted of a large block of PEGylated monoester norbornene and were capped with a short block of norbornene dicarboxylic anhydride. This cap served as a reactive linker that facilitated attachment of the polymer to lysine residues under mildly alkaline conditions. The generality of this approach was shown by synthesizing multivalent polynorbornene-modified viral nanoparticles derived from bacteriophage Qß, a protein nanoparticle used extensively for nanomedicine. The conjugated nanoparticles showed no cytotoxicity to NIH 3T3 murine fibroblast cells. These findings establish protein bioconjugation with functionalized polynorbornenes as an effective alternative to conventional protein/polymer modification strategies and further expand the toolbox for protein bioconjugates.

Coextruded, aligned, and gradient-modified poly(ε-caprolactone) fibers as platforms for neural growth.

Polymeric fibers are of increasing interest to regenerative medicine, as materials made from these fibers are porous, allowing for cell infiltration, influx of nutrients, and efflux of waste products. Recently, multilayered coextrusion has emerged as a scalable and rapid fabrication method to yield microscale to submicron fibers. In this report, we describe the multilayered coextrusion of aligned poly(ε-caprolactone) (PCL) fibers, followed by a simple photochemical patterning to create surface-immobilized gradients onto the polymer fibers. PCL fibers were photochemically decorated with a linear gradient of propargyl benzophenone using a gradient photomask to control light source intensity. The pendant alkynes were then able to undergo the copper-catalyzed azide-alkyne cycloaddition reaction with an azide-modified IKVAV peptide to further functionalize the surface. Gradient-modified IKVAV fibers were evaluated for neural cell adhesion and neural differentiation, using PC-12 cells cultured onto the fibers. The aligned gradient fibers provided directional cues for neurite outgrowth and alignment of neural cells, as observed by cellular elongation, neurite differentiation, and orientation. The work presented herein describes a scalable fiber system combined with simple chemical patterning to generate aligned fibers with controlled surface gradients as cell-seeding scaffolds.

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.

Inter-coat protein loading of active ingredients into Tobacco mild green mosaic virus through partial dissociation and reassembly of the virion.

Chemical pesticide delivery is a fundamental aspect of agriculture. However, the extensive use of pesticides severely endangers the ecosystem because they accumulate on crops, in soil, as well as in drinking and groundwater. New frontiers in nano-engineering have opened the door for precision agriculture. We introduced Tobacco mild green mosaic virus (TMGMV) as a viable delivery platform with a high aspect ratio and favorable soil mobility. In this work, we assess the use of TMGMV as a chemical nanocarrier for agriculturally relevant cargo. While plant viruses are usually portrayed as rigid/solid structures, these are "dynamic materials," and they "breathe" in solution in response to careful adjustment of pH or bathing media [e.g., addition of solvent such as dimethyl sulfoxide (DMSO)]. Through this process, coat proteins (CPs) partially dissociate leading to swelling of the nucleoprotein complexes-allowing for the infusion of active ingredients (AI), such as pesticides [e.g., fluopyram (FLP), clothianidin (CTD), rifampicin (RIF), and ivermectin (IVM)] into the macromolecular structure. We developed a "breathing" method that facilitates inter-coat protein cargo loading, resulting in up to ~ 1000 AIs per virion. This is of significance since in the agricultural setting, there is a need to develop nanoparticle delivery strategies where the AI is not chemically altered, consequently avoiding the need for regulatory and registration processes of new compounds. This work highlights the potential of TMGMV as a pesticide nanocarrier in precision farming applications; the developed methods likely would be applicable to other protein-based nanoparticle systems.

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.

Biocomposite thermoplastic polyurethanes containing evolved bacterial spores as living fillers to facilitate polymer disintegration.

The field of hybrid engineered living materials seeks to pair living organisms with synthetic materials to generate biocomposite materials with augmented function since living systems can provide highly-programmable and complex behavior. Engineered living materials have typically been fabricated using techniques in benign aqueous environments, limiting their application. In this work, biocomposite fabrication is demonstrated in which spores from polymer-degrading bacteria are incorporated into a thermoplastic polyurethane using high-temperature melt extrusion. Bacteria are engineered using adaptive laboratory evolution to improve their heat tolerance to ensure nearly complete cell survivability during manufacturing at 135 °C. Furthermore, the overall tensile properties of spore-filled thermoplastic polyurethanes are substantially improved, resulting in a significant improvement in toughness. The biocomposites facilitate disintegration in compost in the absence of a microbe-rich environment. Finally, embedded spores demonstrate a rationally programmed function, expressing green fluorescent protein. This research provides a scalable method to fabricate advanced biocomposite materials in industrially-compatible processes.