PhotothermalPhage: A Virus-Based Photothermal Therapeutic Agent.

Virus-like particles (VLPs) are multifunctional nanocarriers that mimic the architecture of viruses. They can serve as a safe platform for specific functionalization and immunization, which provides benefits in a wide range of biomedical applications. In this work, a new generation immunophotothermal agent is developed that adjuvants photothermal ablation using a chemically modified VLP called bacteriophage Qß. The design is based on the conjugation of near-infrared absorbing croconium dyes to lysine residues located on the surface of Qß, which turns it to a powerful NIR-absorber called PhotothermalPhage. This system can generate more heat upon 808 nm NIR laser radiation than free dye and possesses a photothermal efficiency comparable to gold nanostructures, yet it is biodegradable and acts as an immunoadjuvant combined with the heat it produces. The synergistic combination of thermal ablation with the mild immunogenicity of the VLP leads to effective suppression of primary tumors, reduced lung metastasis, and increased survival time.

Supramolecular Encapsulation of Small-Ultrared Fluorescent Proteins in Virus-Like Nanoparticles for Noninvasive In Vivo Imaging Agents.

Icosahedral virus-like particles (VLPs) derived from bacteriophages Qß and PP7 encapsulating small-ultrared fluorescent protein (smURFP) were produced using a versatile supramolecular capsid disassemble-reassemble approach. The generated fluorescent VLPs display identical structural properties to their nonfluorescent analogs. Encapsulated smURFP shows indistinguishable photochemical properties to its unencapsulated counterpart, exhibits outstanding stability toward pH, and produces bright in vitro images following phagocytosis by macrophages. In vivo imaging allows the biodistribution to be imaged at different time points. Ex vivo imaging of intravenously administered encapsulated smURFP reveals a localization in the liver and kidneys after 2 h blood circulation and substantial elimination after 16 h of imaging, highlighting the potential application of these constructs as noninvasive in vivo imaging agents.

Protein-Polymer Delivery: Chemistry from the Cold Chain to the Clinic.

Drug delivery is commonly thought of as the performance of a drug in vivo. Rather, the process of drug delivery can comprise of the journey of the drug from manufacturer to clinic, clinic to patient, and patient to disease. Each step of the journey includes hurdles that must be overcome for the therapeutic to be successful. Recent developments in proteinaceous therapeutics have made the successful completion of this journey even more important because of the relatively fragile nature of proteins in a drug delivery context. Polymers have been demonstrated to be an effective complement to proteinaceous therapeutics throughout this journey owing to their flexibility in design and function. During transit from manufacturer to clinic, the proteinaceous drug is threatened by denaturation at elevated temperatures. Polymers can help improve the thermal stability of the drug at ambient shipping conditions, thereby reducing the need for an expensive cold chain to preserve its bioactivity. Upon arrival at the clinic, the drug must be reconstituted into a suitable formulation that can be introduced into the patient. Unfortunately, traditional drug formulations relying on oral administration are generally not suitable for proteinaceous drugs owing to the hostile environment of the stomach. Other traditional methods of drug administration-like hypodermic injections-frequently suffer from low patient compliance. Polymers have been explored to design drug formulations suitable for alternative methods of administration. Upon entry into the body, proteinaceous drugs are at risk for identification, destruction, and excretion by the immune system. Polymers can help drugs reprogram immune system response and, in some cases, elicit a synergistic immune response. The next phase of research on protein-polymer-based therapeutics encourages a holistic effort to design systems that can survive each stage of the drug delivery journey.

A scalable synthesis of adjuvanting antigen depots based on metal-organic frameworks.

Vaccines have saved countless lives by preventing and even irradicating infectious diseases. Commonly used subunit vaccines comprising one or multiple recombinant proteins isolated from a pathogen demonstrate a better safety profile than live or attenuated vaccines. However, the immunogenicity of these vaccines is weak, and therefore, subunit vaccines require a series of doses to achieve sufficient immunity against the pathogen. Here, we show that the biomimetic mineralization of the inert model antigen, ovalbumin (OVA), in zeolitic imidazolate framework-8 (ZIF-8) significantly improves the humoral immune response over three bolus doses of OVA (OVA 3×). Encapsulation of OVA in ZIF-8 (OVA@ZIF) demonstrated higher serum antibody titers against OVA than OVA 3×. OVA@ZIF vaccinated mice displayed higher populations of germinal center (GC) B cells and IgG1+ GC B cells as opposed to OVA 3×, indicative of class-switching recombination. We show that the mechanism of this phenomenon is at least partly owed to the metalloimmunological effects of the zinc metal as well as the sustained release of OVA from the ZIF-8 composite. The system acts as an antigen reservoir for antigen-presenting cells to traffic into the draining lymph node, enhancing the humoral response. Lastly, our model system OVA@ZIF is produced quickly at the gram scale in a laboratory setting, sufficient for up to 20 000 vaccine doses.

Intracellular delivery of virus-like particles using a sheddable linker.

Intracellular targeting is essential for the efficient delivery of drugs and nanotherapeutics. Transporting nanomaterials into cells' cytoplasm for therapeutic purposes can be challenging due to the endosomal trap and lysosomal degradation of cargo. To overcome this issue, we utilized chemical synthesis to design a functional carrier that can escape the endosome and deliver biological materials into the cytoplasm. We synthesized a thiol-sensitive maleimide linker that connects the well-known mitochondria targeting lipophilic triphenylphosphonium cation (TPP) to the surface of a proteinaceous nanoparticle based on the engineered virus-like particle (VLP) Qß. TPP facilitates endosomal escape by its lipophilic and cationic nature, which disrupts the endosomal membrane. Once in the cytosol, glutathione reacts with the thiol-sensitive maleimide linkers, severs the TPP from the nanoparticle, halting its trafficking to the mitochondria, and marooning it in the cytosol. We successfully demonstrated cytosolic delivery of a VLP loaded with Green Fluorescent Protein (GFP) in vitro and small-ultrared fluorescent protein (smURFP) in vivo, where evenly distributed fluorescence is observed in A549 human lung adenocarcinoma cells and the epithelial cells of BALB/c mice lungs. As a proof of concept, we encapsulated luciferase-targeted siRNA (siLuc) inside the VLP decorated with the maleimide-TPP (M-TPP) linker. We observed enhanced luminescence silencing in luciferase-expressing HeLa cells using our sheddable TPP linker compared to control VLPs.

Carrier gas triggered controlled biolistic delivery of DNA and protein therapeutics from metal-organic frameworks.

The efficacy and specificity of protein, DNA, and RNA-based drugs make them popular in the clinic; however, these drugs are often delivered via injection, requiring skilled medical personnel, and producing biohazardous waste. Here, we report an approach that allows for their controlled delivery, affording either a burst or slow release without altering the formulation. We show that when encapsulated within zeolitic-imidazolate framework eight (ZIF-8), the biomolecules are stable in powder formulations and can be inoculated with a low-cost, gas-powered "MOF-Jet" into living animal and plant tissues. Additionally, their release profiles can be modulated through judicious selection of the carrier gas used in the MOF-Jet. Our in vitro and in vivo studies reveal that when CO2 is used, it creates a transient and weakly acidic local environment that causes a near-instantaneous release of the biomolecules through an immediate dissolution of ZIF-8. Conversely, when air is used, ZIF-8 biodegrades slowly, releasing the biomolecules over a week. This is the first example of controlled-biolistic delivery of biomolecules using ZIF-8, which provides a powerful tool for fundamental and applied science research.

Stabilization of supramolecular membrane protein-lipid bilayer assemblies through immobilization in a crystalline exoskeleton.

Artificial native-like lipid bilayer systems constructed from phospholipids assembling into unilamellar liposomes allow the reconstitution of detergent-solubilized transmembrane proteins into supramolecular lipid-protein assemblies called proteoliposomes, which mimic cellular membranes. Stabilization of these complexes remains challenging because of their chemical composition, the hydrophobicity and structural instability of membrane proteins, and the lability of interactions between protein, detergent, and lipids within micelles and lipid bilayers. In this work we demonstrate that metastable lipid, protein-detergent, and protein-lipid supramolecular complexes can be successfully generated and immobilized within zeolitic-imidazole framework (ZIF) to enhance their stability against chemical and physical stressors. Upon immobilization in ZIF bio-composites, blank liposomes, and model transmembrane metal transporters in detergent micelles or embedded in proteoliposomes resist elevated temperatures, exposure to chemical denaturants, aging, and mechanical stresses. Extensive morphological and functional characterization of the assemblies upon exfoliation reveal that all these complexes encapsulated within the framework maintain their native morphology, structure, and activity, which is otherwise lost rapidly without immobilization.

Metal-Organic Framework Encapsulated Whole-Cell Vaccines Enhance Humoral Immunity against Bacterial Infection.

The increasing rate of resistance of bacterial infection against antibiotics requires next generation approaches to fight potential pandemic spread. The development of vaccines against pathogenic bacteria has been difficult owing, in part, to the genetic diversity of bacteria. Hence, there are many potential target antigens and little a priori knowledge of which antigen/s will elicit protective immunity. The painstaking process of selecting appropriate antigens could be avoided with whole-cell bacteria; however, whole-cell formulations typically fail to produce long-term and durable immune responses. These complications are one reason why no vaccine against any type of pathogenic E. coli has been successfully clinically translated. As a proof of principle, we demonstrate a method to enhance the immunogenicity of a model pathogenic E. coli strain by forming a slow releasing depot. The E. coli strain CFT073 was biomimetically mineralized within a metal-organic framework (MOF). This process encapsulates the bacteria within 30 min in water and at ambient temperatures. Vaccination with this formulation substantially enhances antibody production and results in significantly enhanced survival in a mouse model of bacteremia compared to standard inactivated formulations.

Using FRET to measure the time it takes for a cell to destroy a virus.

The emergence of viral nanotechnology over the preceding two decades has created a number of intellectually captivating possible translational applications; however, the in vitro fate of the viral nanoparticles in cells remains an open question. Herein, we investigate the stability and lifetime of virus-like particle (VLP) Qß-a representative and popular VLP for several applications-following cellular uptake. By exploiting the available functional handles on the viral surface, we have orthogonally installed the known FRET pair, FITC and Rhodamine B, to gain insight of the particle's behavior in vitro. Based on these data, we believe VLPs undergo aggregation in addition to the anticipated proteolysis within a few hours of cellular uptake.

Supramolecular and biomacromolecular enhancement of metal-free magnetic resonance imaging contrast agents.

Many contrast agents for magnetic resonance imaging are based on gadolinium, however side effects limit their use in some patients. Organic radical contrast agents (ORCAs) are potential alternatives, but are reduced rapidly in physiological conditions and have low relaxivities as single molecule contrast agents. Herein, we use a supramolecular strategy where cucurbit[8]uril binds with nanomolar affinities to ORCAs and protects them against biological reductants to create a stable radical in vivo. We further overcame the weak contrast by conjugating this complex on the surface of a self-assembled biomacromolecule derived from the tobacco mosaic virus.