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.

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.

Enhanced Stability and Controlled Delivery of MOF-Encapsulated Vaccines and Their Immunogenic Response In Vivo.

Vaccines have an innate tendency to lose their structural conformation upon environmental and chemical stressors. A loss in conformation reduces the therapeutic ability to prevent the spread of a pathogen. Herein, we report an in-depth study of zeolitic imidazolate framework-8 and its ability to provide protection for a model viral vector against denaturing conditions. The immunoassay and spectroscopy analysis together demonstrate enhanced thermal and chemical stability to the conformational structure of the encapsulated viral nanoparticle. The long-term biological activity of this virus-ZIF composite was investigated in animal models to further elucidate the integrity of the encapsulated virus, the biosafety, and immunogenicity of the overall composite. Additionally, histological analysis found no observable tissue damage in the skin or vital organs in mice, following multiple subcutaneous administrations. This study shows that ZIF-based protein composites are strong candidates for improved preservation of proteinaceous drugs, are biocompatible, and are capable of controlling the release and adsorption of drugs in vivo.

ZIF-8 Degrades in Cell Media, Serum, and Some-But Not All-Common Laboratory Buffers.

The emergence of drug delivery using water stable metal-organic frameworks has elicited a lot of interest in their biocompatibility. However, few studies have been conducted on their stability in common buffers, cell media, and blood proteins. For these studies, single crystal ZIF-8 approximately 1 um in diameter were synthesized, incubated with common laboratory buffers, cell media, and serum, and then characterized by PXRD, IR, DLS, and SEM. Time-resolved SEM and PXRD demonstrate that buffers containing phosphate and bicarbonate alter the appearance and composition of ZIF-8; however, cargo inside the ZIF-8 does not appear to leak out, in most of these buffers, even when the ZIF-8 itself is displaced by phosphates. On the other hand, blood proteins in serum dissolve ZIF-8, causing trapped biomolecules to escape. The study presented here suggests that ZIF-8 can undergo dramatic surface chemistry changes that may affect the interpretation of cellular uptake and cargo release data. On the other hand, it provides a rational explanation as to how ZIF-8 neatly dissolves in vivo.

Site-Selective Nucleation and Size Control of Gold Nanoparticle Photothermal Antennae on the Pore Structures of a Virus.

In this Article, we show that the surface of the bacteriophage Qß is equipped with natural ligands for the synthesis of small gold nanoparticles (AuNPs). By exploiting disulfides in the protein secondary structure and the geometry formed from the capsid quaternary structure, we find that we can produce regularly arrayed patterns of ∼6 nm AuNPs across the surface of the virus-like particle. Experimental and computational analyses provide insight into the formation and stability of this composite. We further show that the entrapped genetic material can hold upward of 500 molecules of the anticancer drug Doxorubicin without leaking and without interfering with the synthesis of the AuNPs. This direct nucleation of nanoparticles on the capsid allows for exceptional conduction of photothermal energy upon nanosecond laser irradiation. As a proof of principle, we demonstrate that this energy is capable of rapidly releasing the drug from the capsid without heating the bulk solution, allowing for highly targeted cell killing in vitro.

Regulating the Uptake of Viral Nanoparticles in Macrophage and Cancer Cells via a pH Switch.

Controlling the uptake of nanomaterials into phagocytes is a challenging problem. We describe an approach to inhibit the cellular uptake by macrophages and HeLa cells of nanoparticles derived from bacteriophage Qß by conjugating negatively charged terminal hexanoic acid moieties onto its surface. Additionally, we show hydrazone linkers can be installed between the surface of Qß and the terminal hexanoic acid moieties, resulting in a pH-responsive conjugate that, in acidic conditions, can release the terminal hexanoic acid moiety and allow for the uptake of the Qß nanoparticle. The installation of the "pH switch" did not change the structure-function properties of the hexanoic acid moiety and the uptake of the Qß conjugates by macrophages.

Investigation of Controlled Growth of Metal-Organic Frameworks on Anisotropic Virus Particles.

Biomimetic mineralization with metal-organic frameworks (MOF), typically zeolitic imidazolate framework-8 (ZIF-8), is an emerging strategy to protect sensitive biological substances against denaturing environmental stressors such as heat and proteolytic agents. Additionally, this same biomimetic mineralization process has the potential of being used to create distinct core-shell architectures using genetically or chemically modified viral nanoparticles. Despite the proliferation of examples for ZIF-8 growth on biological or proteinaceous substrates, systematic studies of these processes are few and far between. Herein, we employed the tobacco mosaic virus (TMV) as a model biological template to investigate the biomimetic mineralization of ZIF-8, which has been proven to be a robust MOF for encasing and protecting inlaid biological substances. Our study shows a systematic dependence upon ZIF-8 crystallization parameters, e.g., ligand to metal molar ratio and metal concentration, which can yield several distinct morphologies of TMV@ZIF-8 composites and phases of ZIF-8. Further investigation using charged synthetic conjugates, time dependent growth analysis, and calorimetric analysis has shown that the TMV-Zn interaction plays a pivotal role in the final morphology of the TMV@ZIF-8, which can take the form of either core-shell bionanoparticles or large crystals of ZIF-8 with entrapped TMV located exclusively on the outer facets. The design rules outlined here, it is hoped, will provide guidance in biomimetic mineralization of MOFs on proteinaceous materials using ZIF-8.

Fluorescent Functionalization across Quaternary Structure in a Virus-like Particle.

Proteinaceous nanomaterials and, in particular, virus-like particles (VLPs) have emerged as robust and uniform platforms that are seeing wider use in biomedical research. However, there are a limited number of bioconjugation reactions for functionalizing the capsids, and very few of those involve functionalization across the supramolecular quaternary structure of protein assemblies. In this work, we exploit the recently described dibromomaleimide moiety as part of a bioconjugation strategy on VLP Qß to break and rebridge the exposed and structurally important disulfides in good yields. Not only was the stability of the quaternary structure retained after the reaction, but the newly functionalized particles also became brightly fluorescent and could be tracked in vitro using a commercially available filter set. Consequently, we show that this highly efficient bioconjugation reaction not only introduces a new functional handle "between" the disulfides of VLPs without compromising their thermal stability but also can be used to create a fluorescent probe.