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COVID-19 poses a major threat to global health and socioeconomic structures, and the need for a highly effective, antimicrobial face mask has been considered a major challenge for protection against respiratory diseases. Here, we report the development of a universal, antiviral, and antibacterial material that can be dip-/spray-coated over conventional mask fabrics to exhibit antimicrobial activities. Our data shows that antimicrobial fabrics rapidly inactivated multiple types of viruses, i.e., human (alpha/beta) coronaviruses, the influenza virus, and bacteria, irrespective of their modes of transmission (aerosol or droplet). This research provides an immediate method to contain infectious diseases, such as COVID-19.

Background Despite the extensive endeavours, developing an effective malaria vaccine remains as a great challenge. Apical membrane antigen 1 (AMA-1) located on the merozoite surface of parasites belonging to the genus Plasmodium is involved in red blood cell invasion. Methods Influenza virus-like particle (VLP) vaccines containing codon-optimized or native (non-codon optimized) AMA-1 from Plasmodium berghei were generated. VLP-induced protective immunity was evaluated in a mouse model. Results Mice immunized with VLP vaccine containing the codon-optimized AMA-1 elicited higher levels of P. berghei-specific IgG and IgG2a antibody responses compared to VLPs containing non-codon optimized AMA-1 before and after challenge infection. Codon-optimized AMA-1 VLP vaccination induced higher levels of CD4+ T cells, CD8+ T cells, B cells, and germinal centre cell responses compared to non-codon optimized AMA-1 VLPs. Importantly, the codon-optimized AMA-1 VLP vaccination showed lower body weight loss, longer survival and a significant decrease in parasitaemia compared to non-codon optimized VLP vaccination. Conclusion Overall, VLP vaccine expressing codon-optimized AMA-1 induced better protective efficacy than VLPs expressing the non-codon optimized AMA-1. Current findings highlight the importance of codon-optimization for vaccine use and its potential involvement in future malaria vaccine design strategies.

Curcumin is a polyphenolic compound present in turmeric with extensive uses in cooking foods and biomedical applications. However, due to its hydrophobic nature, it is poorly soluble in water and its bioavailability is very low on oral administration in organisms. In this study, we investigated the dietary curcumin nanospheres in a weaned piglet model based on the growth, serum biochemistry, proteomics, fecal coliform bacteria, and malodors in the feces of piglets. A total of 135 weaned piglets (Duroc × [Yorkshire × Landrace]) with an average initial body weight of 7.0 ± 1.0 kg (28 ± 1 days of age) were randomly distributed in 9 pens (15 pigs in each pen) fed the dietary curcumin nanospheres (CN) at 0 (control), 0.5 (T1), and 1.0 mL (T2) CN/kg of diet in triplicates for 21 days. At the end of the feeding trial, the results showed piglets fed 1.0 mL CN/kg diet had significantly higher growth performance and feed utilization than control diet (without CN). However, there were no significant differences in growth and feed utilization between piglets fed T1 and T2 diets. Serum glucose, gamma-glutamyl transferase, total bilirubin, amylase, and lipase contents were unaffected in piglets fed the experimental diets. Interestingly, piglets fed T1 and T2 diets showed significantly lower total cholesterol levels than control diet. In serum proteomics, a total of 103 differentially expressed proteins (DEPs) were identified in the piglets fed control, T1, and T2 diets, of which 14 DEPs were upregulated and 4 DEPs were downregulated. Fecal coliform bacteria and ammonia gas were significantly reduced in piglets fed T1 and T2 diets. Overall, the results indicated dietary supplementation of CN could enhance the growth, feed utilization, and immunity—and reduce fecal pathogenic bacteria as well as ammonia gas emissions—in weaned piglets.

As COVID-19 exemplifies, respiratory diseases transmitted through aerosols or droplets are global threats to public health, and respiratory protection measures are essential first lines of infection prevention and control. However, common face masks are single use and can cause cross-infection due to the accumulated infectious pathogens. We developed salt-based formulations to coat membrane fibers to fabricate antimicrobial filters. Here, we report a mechanistic study on salt-induced pathogen inactivation. The salt recrystallization following aerosol exposure was characterized over time on sodium chloride (NaCl), potassium sulfate (K2SO4), and potassium chloride (KCl) powders and coatings, which revealed that NaCl and KCl start to recrystallize within 5 min and K2SO4 within 15 min. The inactivation kinetics observed for the H1N1 influenza virus and Klebsiella pneumoniae matched the salt recrystallization well, which was identified as the main destabilizing mechanism. Additionally, the salt-coated filters were prepared with different methods (with and without a vacuum process), which led to salt coatings with different morphologies for diverse applications. Finally, the salt-coated filters caused a loss of pathogen viability independent of transmission mode (aerosols or droplets), against both DI water and artificial saliva suspensions. Overall, these findings increase our understanding of the salt-recrystallization-based technology to develop highly versatile antimicrobial filters.

Point of care (POC) devices enable immediate diagnosis and treatment in settings where equipped facilities, trained expertise and organized transportation are lacking. Epidemics require rapid treatment to deter the spread of diseases. Thus, POC devices can be used to rapidly diagnose the disease, without the need for trained personnel or expensive equipment. Paper is an excellent option for POC applications, mainly due to its physical and chemical properties and cost. Paper has been used in various forms to detect proteins and nucleic acids from pathogens, as they are the most commonly targeted biomolecules for the detection of infectious diseases. Due to their specificity, a diverse range of biomolecules could be detected using methods such as ELISA, gold nanoparticles and electrochemistry. In spite of the aforementioned advantages, instability and cross-reactivity in protein detection and the complexity of nucleic acid detection deter the rapid replacement of conventional diagnostic methods. Thus, in this chapter, we present key technical progress in paper-based POC devices, identify major functional components and provide insight into important challenges faced to produce optimal diagnostic devices.

Respiratory protection is key in infection prevention of airborne diseases, as highlighted by the COVID-19 pandemic for instance. Conventional technologies have several drawbacks (i.e., cross-infection risk, filtration efficiency improvements limited by difficulty in breathing, and no safe reusability), which have yet to be addressed in a single device. Here, we report the development of a filter overcoming the major technical challenges of respiratory protective devices. Large-pore membranes, offering high breathability but low bacteria capture, were functionalized to have a uniform salt layer on the fibers. The salt-functionalized membranes achieved high filtration efficiency as opposed to the bare membrane, with differences of up to 48%, while maintaining high breathability (> 60% increase compared to commercial surgical masks even for the thickest salt filters tested). The salt-functionalized filters quickly killed Gram-positive and Gram-negative bacteria aerosols in vitro, with CFU reductions observed as early as within 5 min, and in vivo by causing structural damage due to salt recrystallization. The salt coatings retained the pathogen inactivation capability at harsh environmental conditions (37 °C and a relative humidity of 70%, 80% and 90%). Combination of these properties in one filter will lead to the production of an effective device, comprehensibly mitigating infection transmission globally.

This chapter discusses the use of biopolymers/natural polymers in sustainable systems to solve current concerns over petroleum-based polymers. A comprehensive understanding and assessment of the current status of traditional biopolymers through the integration of various disciplines, such as engineering, biotechnology, chemistry, and biology, is expected to provide a systematic view on future research directions. Biopolymers have re-emerged into a new era of sustainable materials, and are expected to take an increasingly central role in the development of revolutionary applications. The competitive position of biopolymers can be achieved through technological progress to resolve multiple drawbacks related to materials developments, properties, and production costs.

Microparticles (MPs) with pH-responding macropores have recently proved their significance for the delivery of vulnerable biomolecules for oral drug administration. The previous MP systems were proven to provide enhanced protection against the gastric environment, however, their application is hindered due to insufficient loading efficiencies and deficient penetration capabilities of encapsulated drugs across the mucus barrier. Here, we report a new co-delivery approach based on amine-functionalized halloysite nanotube (HNT)-embedded MPs (amine-HNT-MPs) with pH-responding macropores specifically designed to deal with the mucus barrier at the absorption site. The mean diameter and polydispersity index of the pored MPs were measured by a particle size analyzer to be 37.6 ± 1.3 µm and 1.15, respectively. The drug loading capacity of the co-delivery system was shown to be 50-times higher than previously reported pored MPs. Fluorescence microscopy analysis of sulforhodamine B (into a hollow interior of HNTs)/ fluorescent nanoparticles (into a hollow interior of MPs)-encapsulated MPs confirmed biphasic release behavior due to pH-dependent pore closing/opening in the simulated gastrointestinal (GI) digestive conditions. To verify the protective effect of the co-delivery system, bromelain and lactase were loaded into HNTs and MPs, respectively, and found to exhibit 94.5 ± 3.3% (bromelain) and 70 ± 14.1% (lactase) functional activity in simulated GI tract conditions. The considerable improvement in the stability of the encapsulated enzymes against gastric conditions are attributed to the efficient pore sealing of the co-delivery system after the encapsulation of enzymes and maintenance of these closed pores in the gastric environment. Furthermore, the mucolytic enzyme (i.e. bromelain)-encapsulated co-delivery system was found to enhance mucopenetration of the encapsulated drug from histological analysis using ex vivo porcine intestine tissue. Therefore, the new microencapsulation design proposed in this study provides a promising solution to the major issues hampering the wide-spread application of MPs in the development of oral drug formulations for biopharmaceuticals and vaccines.

Biofilms of methicillin-resistant Staphylococcus aureus (MRSA) have serious clinical implications. However, it is difficult to eradicate MRSA biofilms due to the increased tolerance to antimicrobials of biofilms. In this study, we investigated the synergistic anti-biofilm effect of the combination of octyl gallate (OG), an antioxidant approved by the US Food and Drug Administration (FDA) as a food additive, and bacitracin, an antimicrobial peptide commonly used in topical antimicrobial ointments. The results of biofilm assays showed that OG enabled bacitracin at concentrations as low as 10-3 U/ml to inhibit biofilm formation in MRSA. A confocal microscopic analysis exhibited that the combination of bacitracin and OG suppressed biofilm formation in MRSA highly effectively compared to the single treatment of either bacitracin or OG. The synergistic anti-biofilm activity of bacitracin and OG was also confirmed in MRSA strains from humans, including USA300, which is the predominant clone of community-associated MRSA in the US. To the best of our knowledge, this is the first report about the synergistic anti-biofilm activity of an antimicrobial peptide and an antioxidant against MRSA.

The development of a smart microencapsulation system programmed to actively respond to environmental pH change has long been recognized a key technology in pharmaceutical and food sciences. To this end, we developed hollow microparticles (MPs) with self-controlled macropores that respond to environmental pH change, using an Oil-in-Water emulsion technique, for oral drug delivery. We observed that freeze-drying of MPs induced closure of macropores. The closing/opening behavior of macropores was confirmed by exposing MPs encapsulating different ingredients (sulforhodamine b, fluorescent nanoparticles, and lactase) to simulated gastrointestinal (GI) fluids. MPs maintained their intact, closed pore structure in gastric pH, and subsequent exposure to intestinal pH resulted in pore opening and ingredients release. Further, MPs displayed higher protection (>15 times) than commercial lactase formulation, indicating the protective ability of the system against harsh GI conditions. This study showed development of a hybrid MP system combining the advantages of solid particles and hollow capsules, exhibiting easy solvent-free loading mechanism and smart protection/release of encapsulates through controllable macropores. Ultimately, our MPs system strives to usher a new research area in smart drug delivery systems and advance the current oral drug delivery technology by solving major challenges in targeted delivery of pH-sensitive therapeutics.

It is a high priority to develop a simple and effective delivery method for a cross-protective influenza vaccine. We investigated skin immunization by microneedle (MN) patch with human influenza split vaccine and virus-like particles containing heterologous M2 extracellular (M2e) domains (M2e5x virus-like particles (VLP)) as a cross-protective influenza vaccine candidate. Co-delivery of influenza split vaccine and M2e5x VLP to the skin by MN patch was found to confer effective protection against heterosubtypic influenza virus by preventing weight loss and reducing lung viral loads. Compared to intramuscular immunization, MN-based delivery of combined split vaccine and M2e5x VLPs shaped cellular immune responses toward T helper type 1 responses increasing IgG2a isotype antibodies as well as IFN-&gamma, producing cells in mucosal and systemic sites. This study provides evidence that potential immunological and logistic benefits of M2e5x VLP with human influenza split vaccine delivered by MN patch can be used to develop an easy-to-administer cross-protective influenza vaccine.

Routes of drug administration and the corresponding physicochemical characteristics of a given route play significant roles in therapeutic efficacy and short term/long term biological effects. Each delivery method has favorable aspects and limitations, each requiring a specific delivery vehicles design. Among various routes, oral delivery has been recognized as the most attractive method, mainly due to its potential for solid formulations with long shelf life, sustained delivery, ease of administration and intensified immune response. At the same time, a few challenges exist in oral delivery, which have been the main research focus in the field in the past few years. The present work concisely reviews different administration routes as well as the advantages and disadvantages of each method, highlighting why oral delivery is currently the most promising approach. Subsequently, the present work discusses the main obstacles for oral systems and explains the most recent solutions proposed to deal with each issue.

A painless self-immunization method with effective and broad cross-protection is urgently needed to prevent infections against newly emerging influenza viruses. In this study, we investigated the cross-protection efficacy of trivalent influenza vaccine containing inactivated A/PR/8/34 (H1N1), A/Hong Kong/68 (H3N2) and B/Lee/40 after skin vaccination using microneedle patches coated with this vaccine. Microneedle vaccination of mice in the skin provided 100% protection against lethal challenges with heterologous pandemic strain influenza A/California/04/09, heterogeneous A/Philippines/2/82 and B/Victoria/287 viruses 8 months after boost immunization. Cross-reactive serum IgG antibody responses against heterologous influenza viruses A/California/04/09, A/Philippines/2/82 and B/Victoria/287 were induced at high levels. Hemagglutination inhibition titers were also maintained at high levels against these heterogeneous viruses. Microneedle vaccination induced substantial levels of cross-reactive IgG antibody responses in the lung and cellular immune responses, as well as cross-reactive antibody-secreting plasma cells in the spleen. Viral loads in the lung were significantly (p

Influenza virus M2 protein has a highly conserved ectodomain (M2e) as a cross-protective antigenic target. We investigated the antigenic and immunogenic properties of tandem repeat M2e (5xM2e) proteins and virus-like particles (5xM2e VLP) to better understand how VLP and protein platform vaccines induce innate and protective adaptive immune responses. Despite the high antigenic properties of 5xM2e proteins, the 5xM2e VLP was superior to 5xM2e proteins in inducing IgG2a isotype antibodies, T cell responses, plasma cells and germinal center B cells as well as in conferring cross protection. Mice primed with 5xM2e VLP were found to be highly responsive to 5xM2e protein boost, overcoming the low immunogenicity and protective efficacy of 5xM2e proteins. Immunogenic differences between VLPs and proteins in priming immune responses might be due to an intrinsic ability of 5xM2e VLP to stimulate dendritic cells secreting T helper type 1 (Th1) cytokines. We also found that 5xM2e VLP was effective in inducing inflammatory cytokines and chemokines, and in recruiting macrophages, monocytes, neutrophils, and CD11b+ dendritic cells at the injection site. Therefore, this study provides evidence that 5xM2e VLP is an effective vaccine platform, inducing cross-protection by stimulating innate and adaptive immune responses.

Microparticles (MPs) have been extensively researched as a potential drug delivery vehicle. Here, we investigated the fabrication of MPs with pH-responsive macropores and evaluated their potential applicability in developing solid oral drug formulations. Our previous study showed that macropored MPs, made of Eudragit® L100-55, could encapsulate 100 nm, 1 µm, and 4 µm sized fluorescent beads—model drugs that are mimicking vaccines, bacteria, and cells. In the present study, closed-pored MPs after freeze-drying were coated with a gastric soluble Eudragit® EPO layer to protect MPs in the simulated pregastric environment. Subsequently, drug encapsulated MPs maintained their intact closed-pored structure in the simulated gastric environment and exhibited a rapid release in the simulated intestine environment. Our MP system was found to provide a significantly higher level of protection to the encapsulated lactase enzyme compared to the control sample (i.e. without using MPs). Real-time fluorescence microscopy analysis showed that macropored MPs released encapsulated drugs in a burst-release pattern and in a size-independent manner. This work shows that our proposed EPO-coated MPs with pH-responsive macropores can meet the challenges posed by the multiple physiological environments of the digestive tract and be used in developing highly effective solid oral drug/vaccine formulations.

Graphical abstract, Oral drugs present the most convenient, economical, and painless route for self-administration. Despite commercialization of multiple technologies relying on micro- and nanocrystalline drugs, research on microparticles (MPs) based oral biopharmaceuticals delivery systems has still not culminated well enough in commercial products. This is largely due to the drugs being exposed to the destabilizing environment during MP synthesis process, and partly because of complicated process conditions. Hence, we developed a solvent swelling-evaporation method of producing pH-responsive MPs with micron-sized macropores using poly(methacrylic acid-co-ethyl acrylate) in 1:1 ratio (commercial name: Eudragit® L100-55 polymer). We investigated the effects of temperature and evaporation time on pore formation, freeze-drying induced pore closure, and the release profile of model drugs (fluorescent beads, lactase, and pravastatin sodium) encapsulated MPs in simulated gastrointestinal tract conditions. Encapsulated lactase/pravastatin maintained >60% of their activity due to the preservation of pore closure, which proved the potential of this proof-of-concept microencapsulation system. Importantly, the presence of macropores on MPs can be beneficial for easy drug loading, and solve the problem of bioactivity loss during the conventional MP fabrication-drug encapsulation steps. Therefore, pH-sensing MPs with macropores can contribute to the development of oral drug formulations for a wide variety of drugs and bio-macromolecules, having a various size ranging from genes to micron-sized ingredients with high therapeutic efficacy.

Respiratory protection against airborne pathogens is crucial for pandemic/epidemic preparedness in the context of personal protection, healthcare systems, and governance. We expect that the development of technologies that overcome the existing challenges in current respiratory protective devices will lead to a timely and effective response to the next outbreak.

Aerosolized pathogens are a leading cause of respiratory infection and transmission. Currently used protective measures pose potential risk of primary/secondary infection and transmission. Here, we report the development of a universal, reusable virus deactivation system by functionalization of the main fibrous filtration unit of surgical mask with sodium chloride salt. The salt coating on the fiber surface dissolves upon exposure to virus aerosols and recrystallizes during drying, destroying the pathogens. When tested with tightly sealed sides, salt-coated filters showed remarkably higher filtration efficiency than conventional mask filtration layer, and 100% survival rate was observed in mice infected with virus penetrated through salt-coated filters. Viruses captured on salt-coated filters exhibited rapid infectivity loss compared to gradual decrease on bare filters. Salt-coated filters proved highly effective in deactivating influenza viruses regardless of subtypes and following storage in harsh environmental conditions. Our results can be applied in obtaining a broad-spectrum, airborne pathogen prevention device in preparation for epidemic and pandemic of respiratory diseases.

Toxoplasma gondii (T. gondii) microneme protein 8 (MIC8) represents a novel, functional distinct invasion factor. In this study, we generated virus-like particles (VLPs) targeting Toxoplasma gondii MIC8 for the first time, and investigated the protection against highly virulent RH strain of T. gondii in a mouse model. We found that VLP vaccination induced Toxoplasma gondii-specific IgG and IgG1 antibody responses in the sera. Upon challenge infection with RH strain of T. gondii tachyzoites, vaccinated mice showed a significant increase of both IgG antibodies in sera and IgA antibodies in feces compared to those before challenge, and a rapid expansion of both germinal center B cell (B220(+), GL7(+)) and T cell (CD4(+), CD8(+)) populations. Importantly, intranasally immunized mice showed higher neutralizing antibodies and displayed no proinflammatory cytokine IFN-gamma in the spleen. Mice were completely protected from a lethal challenge infection with the highly virulent T. gondii (RH) showing no body weight loss (100% survival). Our study shows the effective protection against T. gondii infection provided by VLPs containing microneme protein 8 of T. gondii, thus indicating a potential T. gondii vaccine candidate.

Integrative nanobiotechnology utilizes natural ideas and materials for manufacturing nanoscale devices. As living organisms traditionally represent a good model for engineers to learn from, biological components of interest, with optimal functionality, have been used in the creation of biotic/abiotic hybrid devices. As an example, bacteriorhodopsin/F0F1-ATP-synthase-incorporated polymer vesicles provide a model of hybrid protein/artificial synthetic membrane system to perform biological functions. Some potential applications are the construction of intervesicular/intravesicular communications, such as excitable vesicles (EVs), for biocomputer and biomolecular motor-powered nanoelectromechanical systems (NEMS) for nanomedicine. Finally, advanced biotic/abiotic hybrid technology is expected to provide an alternative method to conventional fabrication technology to meet the increasing demands by saving enormous engineering efforts.

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

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 8 weeks 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.

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 4 weeks 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.

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.

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.

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.

We describe our efforts towards constructing a hybrid protein–polymer vesicle device based on the photoactive protein, bacteriorhodopsin (BR), for applications in the area of biosensors and biofuel cells. Successful protein incorporation into biomimetic polymer vesicles is a prerequisite for developing hybrid 'nano–bio' integrated devices. We suggest a systematic procedure for creating energy transducing, protein-incorporating, functional vesicles, based on the morphological ternary diagram. First, we constructed the morphological ternary diagram of the water/ethanol/polymer system with a size distribution of vesicles. The polymer used was an ABA triblock copolymer, PEtOz–PDMS–PEtOz [poly(2-ethyl-2-oxazoline)-b-poly(dimethylsiloxane)-b-poly(2-ethyl-2-oxazoline)]. Second, we incorporated BR in the form of purple membrane (PM) into polymer vesicle membranes under several different conditions, based on the morphological ternary diagram. Generation of electrochemical energy by BR proton pumping was checked by monitoring the pH change in parallel with transmission electron microscope analysis. The morphology of the polymer vesicles changed very little with the addition of PM. This work shows that the morphological ternary diagram provides a systematic method for constructing successful hybrid BR-incorporating biomimetic polymer vesicles.

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.

Hafnium oxide thin films were deposited on p-type (1 0 0) silicon wafers by reactive dc magnetron sputtering. Prior to the deposition of HfO2 films, a thin Hf film was deposited. Sputtered HfO2 thin films deposited at room temperature remain amorphous at T ° C and orthorhombic phases were observed above 650 °C. The monoclinic phase which is a stable HfO2 polymorphic form appeared after annealing above 800 °C. Capacitance equivalent thickness values decreased and leakage characteristics are improved by the Hf interlayer and O2 settlement process. The decrease of accumulation capacitance values upon annealing is due to the growth of an interfacial layer upon post-annealing. The flat band voltage (VFB) shifts negatively due to positive charge generated during post-annealing.

Oral influenza vaccine provides an efficient means of preventing seasonal and pandemic disease. In this work, the stability of envelope-type split influenza vaccine particles in acidic environments has been investigated. Owing to the fact that hyper-osmotic stress can significantly affect lipid assembly of vaccine, osmotic stress-induced morphological change of split vaccine particles, in conjunction with structural change of antigenic proteins, was investigated by the use of stopped-flow light scattering (SFLS), intrinsic fluorescence, transmission electron microscopy (TEM), and hemagglutination assay. Split vaccine particles were found to exhibit a step-wise morphological change in response to osmotic stress due to double-layered wall structure. The presence of hyper-osmotic stress in acidic medium (0.3 osmolarity, pH 2.0) induced a significant level of membrane perturbation as measured by SFLS and TEM, imposing more damage to antigenic proteins on vaccine envelope than can be caused by pH-induced conformational change at acidic iso-osmotic condition. Further supports were provided by the intrinsic fluorescence and hemagglutinin activity measurements. Thus, hyper-osmotic stress becomes an important factor for determining stability of split vaccine particles in acidic medium. These results are useful in better understanding the destabilizing mechanism of split influenza vaccine particles in gastric environment and in designing oral influenza vaccine formulations.

This study examined the cytotoxicity of halloysite nanotubes (HNTs) by investigating physiological responses of Escherichia coli, from cell growth to protein expression. Surfaces of HNTs were modified by amine functionalization (NH2-HNTs) or bovine serum albumin (BSA) coating and their cytotoxicity levels were compared with that of non-modified HNTs (Bare-HNTs). Bare- and NH2-HNTs exhibited accelerated cell death rates at ≥0.5 mg/ml of HNTs. It was also found that concentration as low as 0.01 mg/ml of HNTs exerted significant toxic effects on the bacterial cells. Cellular viability, metabolic activity, and DNA replication all decreased with increasing concentrations of Bare- and NH2-HNTs. In contrast, 0.01 mg/ml of BSA-coated HNTs (BSA-HNTs) coated showed no evidence of cytotoxicity. Even at concentrations ≤0.1 mg/ml, the cytocompatibility of BSA-HNTs was significantly better than those of Bare- and NH2-HNTs, which was confirmed by the observation of (i) the same or similar levels of cell proliferation and cell viability to the control, and (ii) higher levels of metabolic activity and plasmid DNA replication than those of Bare- and NH2-HNTs. In addition, higher ranaspumin-2 protein yield was observed from bacterial culture supplemented with BSA-HNTs (100, 83, and 80 % of yield at 0.01, 0.05, and 0.1 mg/ml, respectively, relative to the control). This work showed that the increase of bacterial cytotoxicity of HNTs correlated well with elevating HNT concentration and that surface modification of HNTs with amine functional group and BSA coating was an effective strategy to reduce cytotoxicity up to 0.1 mg/ml of HNTs.

Aqueous channels of foam represent a simplified, natural bioreactor on the micro-/nano-scale. Previous studies have demonstrated the feasibility and potential application of foams in replicating cellular process in vitro, but no research has been performed to establish a basis for designing stable and biocompatible foam formulations. Our research has been directed specifically to the evaluation of ranaspumin-2 (RSN-2), a frog foam nest protein. The strong surfactant activity of RSN-2 enabled us to produce foams using low protein concentration (1 mg ml(-1)) over a wide pH range (pH ≥ 3). Importantly, the RSN-2 formulation exhibited the best foam stability at a near neutral pH condition, which shows a potential for application to various biosynthesis applications. Model cellular systems such as liposomes and inactivated A/PR/8/34 influenza virus maintained their physicochemical stability and full hemagglutination activity, indicating biocompatibility of RSN-2 with both cellular membranes and proteins both in bulk solution and in foam. Moreover, the addition of RSN-2 did not exert any deteriorative effects on bacterial cell growth kinetics. In contrast, Tween 20, Triton X-100, and BSA did not show satisfactory performance in terms of foamability, foam stability, physicochemcial stability, and biochemical stability. Although our study has been limited to representative formulations composed of only surfactant molecules, a number of unique advantages make RSN-2 a promising candidate for in vitro foam biosynthesis.

Oral immunization using whole inactivated influenza virus vaccine promises an efficient vaccination strategy. While oral vaccination was hampered by harsh gastric environment, a systematic understanding about vaccine destabilization mechanisms was not performed. Here, we investigated the separate and combined effects of temperature, retention time, pH, and osmotic stress on the stability of influenza vaccine by monitoring the time-dependent morphological change using stopped-flow light scattering. When exposed to osmotic stress, clustering of vaccine particles was enhanced in an acidic medium (pH 2.0) at ≥25°C. Fluorescence spectroscopic studies showed that hyper-osmotic stress at pH 2.0 and 37°C caused a considerable increase in conformational change of antigenic proteins compared to that in acidic iso-osmotic medium. A structural integrity of membrane was destroyed upon exposure to hyper-osmotic stress, leading to irreversible morphological change, as observed by undulation in stopped-flow light scattering intensity and transmission electron microscopy. Consistent with these analyses, hemagglutination activity decreased more significantly with an increasing magnitude of hyper-osmotic stress than in the presence of the hypo- and iso-osmotic stresses. This study shows that the magnitude and direction of the osmotic gradient has a substantial impact on the stability of orally administrated influenza vaccine.