Hyo-Jick Choi

Stability of influenza vaccine coated onto microneedles

A microneedle patch coated with vaccine simplifies vaccination by using a patch-based delivery method and targets vaccination to the skin for superior immunogenicity compared to intramuscular injection. Previous studies of microneedles have demonstrated effective vaccination using freshly prepared microneedles, but the issue of long-term vaccine stability has received only limited attention. Here, we studied the long-term stability of microneedles coated with whole inactivated influenza vaccine guided by the hypothesis that crystallization and phase separation of the microneedle coating matrix damages influenza vaccine coated onto microneedles. In vitro studies showed that the vaccine lost stability as measured by hemagglutination activity in proportion to the degree of coating matrix crystallization and phase separation. Transmission electron microscopy similarly showed damaged morphology of the inactivated virus vaccine associated with crystallization. In vivo assessment of immune response and protective efficacy in mice further showed reduced vaccine immunogenicity after influenza vaccination using microneedles with crystallized or phase-separated coatings. This work shows that crystallization and phase separation of the dried coating matrix are important factors affecting long-term stability of influenza vaccine-coated microneedles.

Stability of whole inactivated influenza virus vaccine during coating onto metal microneedles

Immunization using a microneedle patch coated with vaccine offers the promise of simplified vaccination logistics and increased vaccine immunogenicity. This study examined the stability of influenza vaccine during the microneedle coating process, with a focus on the role of coating formulation excipients. Thick, uniform coatings were obtained using coating formulations containing a viscosity enhancer and surfactant, but these formulations retained little functional vaccine hemagglutinin (HA) activity after coating. Vaccine coating in a trehalose-only formulation retained about 40-50% of vaccine activity, which is a significant improvement. The partial viral activity loss observed in the trehalose-only formulation was hypothesized to come from osmotic pressure-induced vaccine destabilization. We found that inclusion of a viscosity enhancer, carboxymethyl cellulose, overcame this effect and retained full vaccine activity on both washed and plasma-cleaned titanium surfaces. The addition of polymeric surfactant, Lutrol® micro 68, to the trehalose formulation generated phase transformations of the vaccine coating, such as crystallization and phase separation, which was correlated to additional vaccine activity loss, especially when coating on hydrophilic, plasma-cleaned titanium. Again, the addition of a viscosity enhancer suppressed the surfactant-induced phase transformations during drying, which was confirmed by in vivo assessment of antibody response and survival rate after immunization in mice. We conclude that trehalose and a viscosity enhancer are beneficial coating excipients, but the inclusion of surfactant is detrimental to vaccine stability.

Synthesis and characterization of nanoscale biomimetic polymer vesicles and polymer membranes for bioelectronic applications

An amphiphilic ABA triblock copolymer was synthesized using poly(2-ethyl-2-oxazoline) (PEtOz) as hydrophilic block [A] and poly(dimethylsiloxane) (PDMS) as hydrophobic block [B]. The cationic ring-opening polymerization of 2-ethyl-2-oxazoline was initiated by benzyl chloride in the presence of NaI. PEtOz–PDMS–PEtOz was characterized in aqueous solution using transmission electron microscopy (TEM). The block copolymers formed vesicles with a [B] block hydrophobic component thickness of 4 nm, which is thin enough for successful reconstitution of proteins. The mean diameters of the vesicles were measured to be in the range of 150–250 nm, with a narrow distribution. The electrochemical properties of planar PEtOz–PDMS–PEtOz membrane films spread across a Teflon aperture were investigated by electrochemical impedance spectroscopy (EIS). The impedance data showed an increase of planar membrane capacitance (CMEM) from 2.58 × 10−7 to 2.71 × 10−7 F cm−2 and a decrease of membrane resistance (RMEM) from 12 to 10.8 Ω cm2. The increase of CMEM over time in buffer solution can be explained by an increase of the dielectric constant as a result of membrane electrolyte incorporation and/or by the formation and growth of defect in the free-standing films. In contrast to the formation of thin-walled vesicles, the spread triblock copolymer formed a free-standing membrane with a 9 nm thickness. Based on the two possible conformations (bridge midblock conformation and loop midblock conformation) that the triblock copolymer can have, we can conclude that PEtOz–PDMS–PEtOz formed 4 nm thick polymer vesicles with intercalated loop midblock structure in aqueous solution, while 9 nm thick free-standing polymer films with bilayer loop midblock conformation or with bridge midblock conformation were formed by aperture spreading.

Effects of different reconstitution procedures on membrane protein activities in proteopolymersomes

The development of membrane protein reconstitution methods in polymersomes is regarded as a major challenge in replicating cellular functions in engineered cellular mimetic systems. We present a solvent-free membrane protein reconstitution method which can be used in polymersomes. To test our method, we reconstructed in vitro proton-powered ATP synthesis using engineered artificial organelles (BR/F0F1-ATP synthase reconstituted proteopolymersomes). We compared the functionality of BR and ATP synthase between two preparation methods. From the difference in the direction of proton pumping and the ATP production profile, it is evident that the relative orientation of BR can be determined by the condition of the proteins (BR monomer, purple membrane), together with the incorporation techniques. As the new procedure eliminates the problem of protein denaturation during incorporation, this research is expected to enhance the potential applications of synthetic membranes in the future fabrication of hybrid protein/polymer systems.

Biosynthesis within a bubble architecture

Sub-cellular compartmentalization is critical to life; it minimizes diffusion effects and enables locally high concentrations of biochemicals for improved reaction kinetics. We demonstrate an example of in vitro biochemical synthesis inside the water channels of foam using engineered artificial organelles (bacteriorhodopsin and F0F1-ATP synthase reconstituted polymer vesicles) as functional units to produce ATP. These results show that the interstitial space of bubbles serves as a metaphor for sub-cellular structure, providing a new platform for both investigating cellular metabolism and the engineering of biofunctional materials and systems.

Microneedle patch delivery to the skin of virus-like particles containing heterologous M2e extracellular domains of influenza virus induces broad heterosubtypic cross-protection.

A broadly cross-protective influenza vaccine that can be administrated by a painless self-immunization method would be a value as a potential universal mass vaccination strategy. This study developed a minimally-invasive microneedle (MN) patch for skin vaccination with virus-like particles containing influenza virus heterologous M2 extracellular (M2e) domains (M2e5x VLPs) as a universal vaccine candidate without adjuvants. The stability of M2e5x VLP-coated microneedles was maintained for 8weeks at room temperature without losing M2e antigenicity and immunogenicity. MN skin immunization induced strong humoral and mucosal M2e antibody responses and conferred cross-protection against heterosubtypic H1N1, H3N2, and H5N1 influenza virus challenges. In addition, M2e5x VLP MN skin vaccination induced T-helper type 1 responses such as IgG2a isotype antibodies and IFN-γ producing cells at higher levels than those by conventional intramuscular injection. These potential immunological and logistic advantages for skin delivery of M2e5x VLP MN vaccines could offer a promising approach to develop an easy-to-administer universal influenza vaccine.

Light-Driven Hybrid Bioreactor Based on Protein-Incorporated Polymer Vesicles

In vitro biochemical synthesis is regarded as a major challenge in replicating cellular functions in engineered systems. Presented is a nanosized hybrid factory where photo-induced biochemical reactions take place resulting in the production of biomolecules. For this purpose, we reconstructed in vitro proton-powered ATP synthesis using artificial organelles, BR/F0F1-ATP synthase reconstituted polymer vesicles (proteopolymersomes), which have been made without organic solvent. Importantly, BR/F0F1-ATP synthase incorporated polymer vesicles showed an excellent proton leakage characteristic under modified conditions. This research is expected to enhance the potential applications of synthetic artificial membranes from in vitro investigation of cellular metabolism to the fabrication of light-driven biomolecular electronic devices such as optical memory and biofuel cell

Recent Progress in Advanced Nanobiological Materials for Energy and Environmental Applications

In this review, we briefly introduce our efforts to reconstruct cellular life processes by mimicking natural systems and the applications of these systems to energy and environmental problems. Functional units of in vitro cellular life processes are based on the fabrication of artificial organelles using protein-incorporated polymersomes and the creation of bioreactors. This concept of an artificial organelle originates from the first synthesis of poly(siloxane)-poly(alkyloxazoline) block copolymers three decades ago and the first demonstration of protein activity in the polymer membrane a decade ago. The increased value of biomimetic polymers results from many research efforts to find new applications such as functionally active membranes and a biochemical-producing polymersome. At the same time, foam research has advanced to the point that biomolecules can be efficiently produced in the aqueous channels of foam. Ongoing research includes replication of complex biological processes, such as an artificial Calvin cycle for application in biofuel and specialty chemical production, and carbon dioxide sequestration. We believe that the development of optimally designed biomimetic polymers and stable/biocompatible bioreactors would contribute to the realization of the benefits of biomimetic systems. Thus, this paper seeks to review previous research efforts, examine current knowledge/key technical parameters, and identify technical challenges ahead.

Effect of Osmotic Pressure on the Stability of Whole Inactivated Influenza Vaccine for Coating on Microneedles

Enveloped virus vaccines can be damaged by high osmotic strength solutions, such as those used to protect the vaccine antigen during drying, which contain high concentrations of sugars. We therefore studied shrinkage and activity loss of whole inactivated influenza virus in hyperosmotic solutions and used those findings to improve vaccine coating of microneedle patches for influenza vaccination. Using stopped-flow light scattering analysis, we found that the virus underwent an initial shrinkage on the order of 10% by volume within 5 s upon exposure to a hyperosmotic stress difference of 217 milliosmolarity. During this shrinkage, the virus envelope had very low osmotic water permeability (1 – 6×10−4 cm s–1) and high Arrhenius activation energy (E a = 15.0 kcal mol–1), indicating that the water molecules diffused through the viral lipid membranes. After a quasi-stable state of approximately 20 s to 2 min, depending on the species and hypertonic osmotic strength difference of disaccharides, there was a second phase of viral shrinkage. At the highest osmotic strengths, this led to an undulating light scattering profile that appeared to be related to perturbation of the viral envelope resulting in loss of virus activity, as determined by in vitro hemagglutination measurements and in vivo immunogenicity studies in mice. Addition of carboxymethyl cellulose effectively prevented vaccine activity loss in vitro and in vivo, believed to be due to increasing the viscosity of concentrated sugar solution and thereby reducing osmotic stress during coating of microneedles. These results suggest that hyperosmotic solutions can cause biphasic shrinkage of whole inactivated influenza virus which can damage vaccine activity at high osmotic strength and that addition of a viscosity enhancer to the vaccine coating solution can prevent osmotically driven damage and thereby enable preparation of stable microneedle coating formulations for vaccination.

Protein transfer-mediated surface engineering to adjuvantate virus-like nanoparticles for enhanced anti-viral immune responses

UNLABELLED Recombinant virus-like nanoparticles (VLPs) are a promising nanoparticle platform to develop safe vaccines for many viruses. Herein, we describe a novel and rapid protein transfer process to enhance the potency of enveloped VLPs by decorating influenza VLPs with exogenously added glycosylphosphatidylinositol-anchored immunostimulatory molecules (GPI-ISMs). With protein transfer, the level of GPI-ISM incorporation onto VLPs is controllable by varying incubation time and concentration of GPI-ISMs added. ISM incorporation was dependent upon the presence of a GPI-anchor and incorporated proteins were stable and functional for at least 4weeks when stored at 4°C. Vaccinating mice with GPI-granulocyte macrophage colony-stimulating factor (GM-CSF)-incorporated-VLPs induced stronger antibody responses and better protection against a heterologous influenza virus challenge than unmodified VLPs. Thus, VLPs can be enriched with ISMs by protein transfer to increase the potency and breadth of the immune response, which has implications in developing effective nanoparticle-based vaccines against a broad spectrum of enveloped viruses. FROM THE CLINICAL EDITOR The inherent problem with current influenza vaccines is that they do not generate effective cross-protection against heterologous viral strains. In this article, the authors described the development of virus-like nanoparticles (VLPs) as influenza vaccines with enhanced efficacy for cross-protection, due to an easy protein transfer modification process.