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Spherical polyhydroxyalkanoate (PHA) inclusions are naturally self-assembled inside bacteria. These PHA beads are shell-core structures composed of a hydrophobic PHA core surrounded by proteins, such as PHA synthase (PhaC). PhaC is covalently attached and serves as an anchor protein for foreign protein vaccine candidate antigens. PHA beads displaying single and multiple antigens showed enhanced immunological properties when compared to respective soluble vaccines. This review highlights the unique design space of the PHA bead-based vaccines toward the development of safe and synthetic particulate vaccines. The PHA bead technology will be compared with chemically synthesized polyesters, such as polylactic acids, formulated to deliver vaccine antigens. The performance of PHA bead vaccine candidates to induce specific immune responses and protective immunity against bacterial and viral pathogens in animal trials will be summarized. We propose that the PHA bead technology offers a versatile vaccine platform for design and cost-effective manufacture of synthetic multivalent vaccines.

Electrohydrodynamic atomization has been emerging as a powerful approach for respiratory treatment, including the generation and delivery of micro/nanoparticles as carriers for drugs and antigens. In this work, we present a new conceptual design in which two nozzles facilitate dual electrospray coexisting with ionic wind at chamfered tips by a direct current power source. Experimental results by a prototype have demonstrated the capability of simultaneously generating-and-delivering a stream of charged reduced particles. The concept can be beneficial to pulmonary nano-medicine delivery since the mist of nanoparticles is migrated without any restriction of either the collector or the assistance of external flow, but is pretty simple in designing and manufacturing devices.

Cadmium (Cd)-contaminated waterbodies are a worldwide concern for the environment, impacting human health. To address the need for efficient, sustainable and cost-effective remediation measures, we developed innovative Cd bioremediation agents by engineering Escherichia coli to assemble poly(3-hydroxybutyric acid) (PHB) beads densely coated with Cd-binding peptides. This was accomplished by translational fusion of Cd-binding peptides to the N- or C-terminus of a PHB synthase that catalyzes PHB synthesis and mediates assembly of Cd2 or Cd1 coated PHB beads, respectively. Cd1 beads showed greater Cd adsorption with 441 nmol Cd mg−1 bead mass when compared to Cd2 beads (334 nmol Cd mg−1 bead-mass) and plain beads (238 nmol Cd mg−1 bead-mass). The Cd beads were not ecotoxic and did attenuate Cd-spiked solutions toxicity. Overall, the bioengineered beads provide a means to remediate Cd-contaminated sites, can be cost-effectively produced at large scale, and offer a biodegradable and safe alternative to synthetic ecotoxic treatments.

Subunit vaccines are more advantageous than live attenuated vaccines in terms of safety and scale-up manufacture. However, this often comes as a trade-off to their efficacy. Over the years, polymeric nanoparticles have been developed to improve vaccine potency, by engineering their physicochemical properties to incorporate multiple immunological cues to mimic pathogenic microbes and viruses. This review covers recent advances in polymeric nanostructures developed toward particulate vaccines. It focuses on the impact of microbe mimicry (e.g. size, charge, hydrophobicity, and surface chemistry) on modulation of the nanoparticles’ delivery, trafficking, and targeting antigen-presenting cells to elicit potent humoral and cellular immune responses. This review also provides up-to-date progresses on rational designs of a wide variety of polymeric nanostructures that are loaded with antigens and immunostimulatory molecules, ranging from particles, micelles, nanogels, and polymersomes to advanced core-shell structures where polymeric particles are coated with lipids, cell membranes, or proteins., Graphical abstract Image 1

The deformation of soft nanocapsules during cell binding and endocytosis leads to a lower cellular uptake than the stiff ones., The ability of cells to sense external mechanical cues is essential for their adaptation to the surrounding microenvironment. However, how nanoparticle mechanical properties affect cell-nanoparticle interactions remains largely unknown. Here, we synthesized a library of silica nanocapsules (SNCs) with a wide range of elasticity (Young’s modulus ranging from 560 kPa to 1.18 GPa), demonstrating the impact of SNC elasticity on SNC interactions with cells. Transmission electron microscopy revealed that the stiff SNCs remained spherical during cellular uptake. The soft SNCs, however, were deformed by forces originating from the specific ligand-receptor interaction and membrane wrapping, which reduced their cellular binding and endocytosis rate. This work demonstrates the crucial role of the elasticity of nanoparticles in modulating their macrophage uptake and receptor-mediated cancer cell uptake, which may shed light on the design of drug delivery vectors with higher efficiency.

Encapsulation of biomoleucles in metal organic frameworks (MOFs) has recently attracted significant interest because of the benign process including room temperature, neutral pH and without the requirement of any other chemical reagents. Also, these biomolecule incorporated MOFs (biomolecules@MOFs) have demonstrated their potential in biomolecule protection and controlled release for various applications such as drug delivery, vaccines, etc. This work aims to develop a general strategy to make biomolecules@MOFs via a biomimetic mineralization process.

Encapsulation of biomoleucles in metal organic frameworks (MOFs) has recently attracted significant interest because of the benign process including room temperature, neutral pH and without the requirement of any other chemical reagents. Also, these biomolecule incorporated MOFs (biomolecules@MOFs) have demonstrated their potential in biomolecule protection and controlled release for various applications such as drug delivery, vaccines, etc. This work aims to develop a general strategy to make biomolecules@MOFs via a biomimetic mineralization process.

Antibiotic resistance poses a growing threat to global public health. It is urgent to develop new alternative antibiotics. Antimicrobial peptide (AMP) is a diverse class of natural-occurring molecules that constitute immune systems of living organisms. More than 2500 AMPs have been identified and isolated from natural sources. Compared to conventional antibiotics, AMPs exhibit antimicrobial activities against a broad spectrum of microorganisms including bacteria, fungi, and even viruses. More importantly, the unique antimicrobial mechanisms of AMPs make it difficult for microorganisms to develop resistance. Therefore, it is very promising to develop AMPs as high-value antimicrobial candidates. This mini review provides an update of recent progresses in recombinant production of AMPs after fusion of AMP with carrier proteins and their scale-up. Key factors including selection of expression host and fusion tags are firstly introduced, followed by subsequent discussions on purification of fusion proteins and recovery of antimicrobial peptides. The scale production of AMPs is also explored.

Precise engineering of nanoparticles with systematically varied properties (size, charge surface properties, targeting ligands, etc.) remains a challenge, limiting the effective optimization of nanoparticles for particular applications. Herein we report a single-step microfluidic combinatorial approach for producing a library of single and dual-ligand liposomes with systematically-varied properties including size, zeta potential, targeting ligand, ligand density, and ligand ratio. A targeting ligand folic acid and a cell penetrating peptide TAT were employed to achieve the optimal synergistic targeting effect. In 2D cell monolayer models, the single-ligand folic acid modified liposome didn’t show any enhanced cellular uptake, while the incorporation of TAT peptide “switched on” the function of folic acid, and induced significant elevated cellular uptake compared to the single ligand modified liposomes, showing a strong synergistic targeting effect. The folic acid and TAT peptide dual-ligand liposome also demonstrated enhanced tumor penetration as observed using 3D tumor spheroid models. The in vivo study further confirmed the improved tumor targeting and longer tumor retention (up to 72 h) of the dual-ligand liposomes. Our work not only proved the versatility of this microfluidic combinatorial approach in producing libraries of multifunctional liposomes with controlled properties but also revealed the great potential of the optimized liposome formulation for synergistic targeting effects.

In recent years, antimicrobial peptides (AMPs) have attracted increasing attention. The microbial cells provide a simple, cost-effective platform to produce AMPs in industrial quantities. While AMP production as fusion proteins in microorganisms is commonly used, the recovery of AMPs necessitates the use of expensive proteases and extra purification steps. Here, we develop a novel fusion protein DAMP4-F-pexiganan comprising a carrier protein DAMP4 linked to the AMP, pexiganan, through a long, flexible linker. We show that this fusion protein can be purified using a non-chromatography approach and exhibits the same antimicrobial activity as the chemically synthesized pexiganan peptide without any cleavage step. Activity of the fusion protein is dependent on a long, flexible linker between the AMP and carrier domains, as well as on the expression conditions of the fusion protein, with low-temperature expression promoting better folding of the AMP domain. The production of DAMP4-F-pexiganan circumvents the time-consuming and costly steps of chromatography-based purification and enzymatic cleavages, therefore shows considerable advantages over traditional microbial production of AMPs. We expect this novel fusion protein, and the studies on the effect of linker and expression conditions on its antimicrobial activity, will broaden the rational design and production of antimicrobial products based on AMPs.

The physicochemical properties of nanoparticles (size, charge, and surface chemistry, etc.) influence their biological functions often in complex and poorly understood ways. This complexity is compounded when the nanostructures involved have variable mechanical properties. Here, we report the synthesis of liquid-filled silica nanocapsules (SNCs, ∼ 150 nm) having a wide range of stiffness (with Young's moduli ranging from 704 kPa to 9.7 GPa). We demonstrate a complex trade-off between nanoparticle stiffness and the efficiencies of both immune evasion and passive/active tumor targeting. Soft SNCs showed 3 times less uptake by macrophages than stiff SNCs, while the uptake of PEGylated SNCs by cancer cells was independent of stiffness. In addition, the functionalization of stiff SNCs with folic acid significantly enhanced their receptor-mediated cellular uptake, whereas little improvement for the soft SNCs was conferred. Further in vivo experiments confirmed these findings and demonstrated the critical role of nanoparticle mechanical properties in regulating their interactions with biological systems.

Multiphase microfluidics has attracted significant interest in making micro- and nanostructures for various applications because of its capabilities in precisely controlling and manipulating a small volume of liquids. In this review, we introduce the recent advances in making micro- and nanostructures for pharmaceutical applications, including microparticles and microcapsules for controlled release, nanoparticles for drug delivery and microgels for 3D cell culture. With the development of more advanced microfluidic systems, the research focus in this field has shifted from making simple micro- and nanostructures to multifunctional systems to achieve more desirable functions. However, these multifunctions may lose their advantages that have been demonstrated in vitro once they are applied in vivo or later in human. The key challenge is a lack of fundamental understanding of the interactions between the micro- and nanomaterials and the biology systems. Consequently, the translation of these advanced materials lags far behind their extensive laboratory research. To better understand the micro- or nano-bio interactions, the development of new in vivo-mimicking models is imperative. Microfluidics has demonstrated its great potential in creating physiologically relevant models including 3D cell culture, tumor-on-a-chip and organs-on-a-chip. Therefore, efforts towards developing 3D cell culture and biomimetic chips including tumor-on-a-chip and organs-on-chips for faster and reliable evaluation of these micro- and nanosystems are also highlighted.

This paper reports the dynamic interfacial behavior of a new interfacially active peptide AM-S, which was designed based on a peptide surfactant AM1 modularized with an additional silk-derived hydrophobic tail to enhance anchoring to air–water interfaces. AM-S peptide shows a random coil conformation in bulk solution similar to AM1 as determined by circular dichroism spectroscopy, which facilitates rapid adsorption at the air–water interface, reducing interfacial tension from 72 to 52 mN/m within 300 s at a low concentration of 10 μM. Although the interfacial films formed by AM-S demonstrated low tensile stress as compared to AM1, the AM-S films in the presence of Zn(II), but not in its absence, show significant resistance against compression, as peptides were unable to desorb quickly under the compression conditions imposed by the Cambridge interfacial tensiometer (CIT). These results indicate that AM-S peptides tend to undergo a multilayer adsorption at the interfaces, in contrast to AM1 peptide that on...

The physicochemical properties of nanoparticles play critical roles in regulating nano-bio interactions. Whereas the effects of the size, shape, and surface charge of nanoparticles on their biological performances have been extensively investigated, the roles of nanoparticle mechanical properties in drug delivery, which have only been recognized recently, remain the least explored. This review article provides an overview of the impacts of nanoparticle mechanical properties on cancer drug delivery, including (1) basic terminologies of the mechanical properties of nanoparticles and techniques for characterizing these properties; (2) current methods for fabricating nanoparticles with tunable mechanical properties; (3) in vitro and in vivo studies that highlight key biological performances of stiff and soft nanoparticles, including blood circulation, tumor or tissue targeting, tumor penetration, and cancer cell internalization, with a special emphasis on the underlying mechanisms that control those complicated nano-bio interactions at the cellular, tissue, and organ levels. The interesting research and findings discussed in this review article will offer the research community a better understanding of how this research field evolved during the past years and provide some general guidance on how to design and explore the effects of nanoparticle mechanical properties on nano-bio interactions. These fundamental understandings, will in turn, improve our ability to design better nanoparticles for enhanced drug delivery.

A large range of nanoparticles have been developed to encapsulate hydrophobic drugs. However, drug loading is usually less than 10 % or even 1 %. Now, core-shell nanoparticles are fabricated having exceptionally high drug loading up to 65 % (drug weight/the total weight of drug-loaded nanoparticles) and high encapsulation efficiencies (>99 %) based on modular biomolecule templating. Bifunctional amphiphilic peptides are designed to not only stabilize hydrophobic drug nanoparticles but also induce biosilicification at the nanodrug particle surface thus forming drug-core silica-shell nanocomposites. This platform technology is highly versatile for encapsulating various hydrophobic cargos. Furthermore, the high drug loading nanoparticles lead to better in vitro cytotoxic effects and in vivo suppression of tumor growth, highlighting the significance of using high drug-loading nanoparticles.

Various pesticide nanocarriers have been developed. However, their pest-control applications remain limited in laboratories. Herein, we developed silica nanocapsules encapsulating fipronil (SNC) and their engineered form, poly(ethyleneimine)-coated SNC (SNC-PEI), based on recombinant catalytic modular protein D4S2 and used them against termite colonies Coptotermes lacteus in fields. To achieve this, an integrated biomolecular bioprocess was developed to produce D4S2 for manufacturing SNC containing fipronil with high encapsulation efficiency of approximately 97% at benign reaction conditions and at scales sufficient for the field applications. PEI coating was achieved via electrostatic interactions to yield SNC-PEI with a slower release of fipronil than SNC without coating. As a proof-of-concept, bait toxicants containing varied fipronil concentrations were formulated and exposed to nine termite mounds, aiming to prolong fipronil release hence allowing sufficient time for termites to relocate the baits into and distribute throughout the colony, and to eliminate that colony. Some baits were relocated into the mounds, but colonies were not eliminated due to several reasons. We caution others interested in producing bait toxicants to be aware of the multilevel resistance mechanisms of the Coptotermes spp. "superorganism".

Microfluidics has been an active research field and an indispensable tool for quantitative analyses of chemicals and biological specimens for decades. Its widespread application stems from its ability to accurately handle small quantities of samples and its well-controlled environments. Further developments of microfluidics require a fundamental understanding of fluid dynamics and associated phenomena. This chapter provides some basic descriptions of fluid properties and principles that govern the flows inside the channels of a microfluidic device. It also examines relevant dimensionless numbers used for the understanding of the flow and the design and scale-up of microfluidic devices. The chapter concludes with suggestions on key areas of importance for future research in microfluidics.

Silica nanocapsules have attracted significant interest due to their core-shell hierarchical structure. The core domain allows the encapsulation of various functional components such as drugs, fluorescent and magnetic nanoparticles for applications in drug delivery, imaging and sensing, and the silica shell with its unique properties including biocompatibility, chemical and physical stability, and surface-chemistry tailorability provides a protection layer for the encapsulated cargo. Therefore, significant effort has been directed to synthesize silica nanocapsules with engineered properties, including size, composition and surface functionality, for various applications. This review provides a comprehensive overview of emerging methods for the manufacture of silica nanocapsules, with a special emphasis on different interfacial engineering strategies. The review starts with an introduction of various manufacturing approaches of silica nanocapsules highlighting surface engineering of the core template nanomaterials (solid nanoparticles, liquid droplets, and gas bubbles) using chemicals or biomolecules which are able to direct nucleation and growth of silica at the boundary of two-phase interfaces (solid-liquid, liquid-liquid, and gas-liquid). Next, surface functionalization of silica nanocapsules is presented. Furthermore, strategies and challenges of encapsulating active molecules (pre-loading and post-loading approaches) in these capsular systems are critically discussed. Finally, applications of silica nanocapsules in controlled release, imaging, and theranostics are reviewed.

Pseudomonas aeruginosa is an opportunistic human pathogen causing devastating acute and chronic infections in individuals with compromised immune systems. Its highly notorious persistence in clinical settings is attributed to its ability to form antibiotic-resistant biofilms. Biofilm is an architecture built mostly by autogenic extracellular polymeric substances which function as a scaffold to encase the bacteria together on surfaces, and to protect them from environmental stresses, impedes phagocytosis and thereby conferring the capacity for colonization and long-term persistence. Here we review the current knowledge on P. aeruginosa biofilms, its development stages, and molecular mechanisms of invasion and persistence conferred by biofilms. Explosive cell lysis within bacterial biofilm to produce essential communal materials, and interspecies biofilms of P. aeruginosa and commensal Streptococcus which impedes P. aeruginosa virulence and possibly improves disease conditions will also be discussed. Recent research on diagnostics of P. aeruginosa infections will be investigated. Finally, therapeutic strategies for the treatment of P. aeruginosa biofilms along with their advantages and limitations will be compiled.

Biomineralization of metal organic frameworks (MOFs) using biomolecules has recently attracted significant interest because of the benign process including room temperature, neutral pH and without the requirement of any other chemical reagents. Also, these biomolecule incorporated MOFs (biomolecules@MOFs) have demonstrated their potential in biomolecule encapsulation, protection and controlled release. This work aims to develop a general strategy to make biomolecules@MOFs via a biomimetic mineralization process. A library of biomolecules (peptides and proteins) with different charges were systematically studied to fundamentally understand the role of biomolecules and their proprieties in biomolecule-mediated MOF biomineralization. Biomolecule charge, amino acid sequence and stirring speed have been demonstrated to play important roles in controlling biomineralization reaction rate, particle shape and morphology.

The concept of dual-ligand targeting has been around for quite some time, but remains controversial due to the intricate interplay between so many different factors such as the choice of dual ligands, their densities, ratios and length matching, etc. Herein, the synthesis of a combinatorial library of single and dual-ligand nanoparticles with systematically varied properties (ligand densities, ligand ratios, and lengths) for tumor targeting is reported. Folic acid (FA) and hyaluronic acid (HA) are used as two model targeting ligands. It is found that the length matching and ligand ratio play critical roles in achieving the synergetic effect of the dual-ligand targeting. When FA is presented on the nanoparticle surface through a 5K polyethylene glycol (PEG) chain, the dual ligand formulations using the HA with either 5K or 10K length do not show any targeting effect, but the right length of HA (7K) with a careful selection of the right ligand ratio do enhance the targeting efficiency and specificity significantly. Further in vitro 3D tumor spheroid models and in vivo xenograft mice models confirm the synergetic targeting efficiency of the optimal dual-ligand formulation (5F2H7K ). This work provides a valuable insight into the design of dual-ligand targeting nanosystems.

Antimicrobial peptides (AMPs) have significant potential as alternatives to classical antibiotics. However, AMPs are currently prepared using processes which are often laborious, expensive and of low-yield, thus hindering their research and application. Large-scale methods for production of AMPs using a cost-effective approach is urgently required. In this study, we report a scalable, chromatography-free downstream processing method for producing an antimicrobial peptide, pexiganan, using recombinant Escherichia coli (E. coli). The four helix bundle structure of the unique carrier protein DAMP4 was used to facilitate a simple and cheap purification process based on a selective thermochemical precipitation. Highly pure fusion protein DAMP4var-pexiganan was obtained at high yield (around 24 mg per 800 mL cell culture with a final cultivation OD600 ~ 2). The purification yield of DAMP4var-pexiganan protein is increased twofold with a 72.9% of the protein recovery in this study as compared to the previous purification processes (Dwyer in Chem Eng Sci 105:12–21, 2014). The antimicrobial peptide pexiganan was released and activated from the fusion protein by a simple acid-cleavage. Isoelectric precipitation was then applied to separate the pexiganan peptide from the DAMP4var protein carrier. The final yield of pure bio-produced pexiganan was 1.6 mg from 800 mL of bacterial cell culture (final cultivation OD600 ~ 2). The minimum bactericidal concentration (MBC) test demonstrated that the bio-produced pexiganan has the same antimicrobial activity as chemically synthesized counterpart. This novel downstream process provides a new strategy for simple and probable economic production of antimicrobial peptides. Electronic supplementary material The online version of this article (10.1186/s13568-018-0541-3) contains supplementary material, which is available to authorized users.

A pesticide delivery system made of biocompatible components and having sustained release properties is highly desirable for agricultural applications. In this study, we report a new biocompatible oil-core silica-shell nanocapsule for sustained release of fipronil insecticide. Silica nanocapsules were prepared by a recently reported emulsion and biomimetic dual-templating approach under benign conditions and without using any toxic chemicals. The loading of fipronil was achieved by direct dissolution in the oil core prior to biomimetic growth of a layer of silica shell surrounding the core, with encapsulation efficiency as high as 73%. Sustained release of fipronil in vitro was tunable through control of the silica-shell thickness (i.e., 8-44 nm). In vivo laboratory tests showed that the insecticidal effect of the fipronil-encapsulated silica nanocapsules against economically important subterranean termites could be controlled by tuning the shell thickness. These studies demonstrated the effectiveness and tunability of an environmentally friendly sustained release system for insecticide, which has great potential for broader agricultural applications with minimal environmental risks.

Designed peptide surfactants offer a number of advanced properties over conventional petrochemical surfactants, including biocompatibility, sustainability, and tailorability of the chemical and physical properties through peptide design. Their biocompatibility and degradability make them attractive for various applications, particularly for food and pharmaceutical applications. In this work, two new peptide surfactants derived from an amphiphilic peptide surfactant (AM1) were designed (AM-S and C8-AM) to better understand links between structure, interfacial activity, and emulsification. Based on AM1, which has an interfacial α-helical structure, AM-S and C8-AM were designed to have two modules, that is, the α-helical AM1 module and an additional hydrophobic moiety to provide for better anchoring at the oil–water interface. Both AM-S and C8-AM at low bulk concentration of 20 μM were able to adsorb rapidly at the oil–water interface and reduced interfacial tension to equilibrium values of 17.0 and 8.4 mN/m...

Silica nanocapsules have attracted tremendous interest for encapsulation, protection, and controlled release of various cargoes due to their unique hierarchical core-shell structure. However, it remains challenging to synthesize silica nanocapsules having high cargo-loading capacity and cargo-protection capability without compromising process simplicity and biocompatibility properties. Here, we synthesized oil-core silica-shell nanocapsules under environmentally friendly conditions by a novel emulsion and biomimetic dual-templating approach using a dual-functional protein, in lieu of petrochemical surfactants, thus avoiding the necessities for the removal of toxic components. A light- and pH-sensitive compound can be facilely encapsulated in the silica nanocapsules with the encapsulation efficiency of nearly 100%. Release of the encapsulated active from the nanocapsules was not shown an indication of undesired burst release. Instead, the release can be tuned by controlling the silica-shell thicknesses (i.e., 40 and 77 nm from which the cargo released at 42.0 and 31.3% of the initial amount after 32 days, respectively). The release kinetics were fitted well to the Higuchi model, enabling the possibility of the prediction of release kinetics as a function of shell thickness, thus achieving design-for-purpose silica nanocapsules. Furthermore, the nanocapsules showed excellent alkaline- and sunlight-shielding protective efficacies, which resulted in significantly prolonged half-life of the sensitive cargo. Our biomimetic silica nanocapsules provide a nanocarrier platform for applications that demand process scalability, sustainability, and biocompatibility coupled with unique cargo-protection and controlled-release properties.

Anaplasma marginale is a devastating tick-borne pathogen causing anaplasmosis in cattle and results in significant economic loss to the cattle industry worldwide. Currently, there is no widely accepted vaccine against A. marginale. New generation subunit vaccines against A. marginale, which are much safer, more efficient and cost-effective, are in great need. The A. marginale outer membrane protein VirB9-1 is a promising antigen for vaccination. We previously have shown that soluble recombinant VirB9-1 protein can be expressed and purified from Escherichia coli and induce a high level of humoral and cellular immunity in mice. In this study, we re-formulated the nanovaccines using the partially-purified VirB9-1 protein as the antigen and hollow nano-size silica vesicles (SV-100) as the adjuvant. We simplified the purification method to obtain the partially-purified antigen VirB9-1 with a six-fold higher yield. The new formulations using the partially-purified VirB9-1 protein achieved higher antibody and cell-mediated immune responses compared to the purified ones. This finding suggests that the partially-purified VirB9-1 protein performs better than the purified ones in the vaccination against A. marginale, and a certain level of contaminants in the protein antigen can be self-adjuvant and boost immunogenicity together with the nanoparticle adjuvant. This may lead to finding a “Goldilocks” level of contaminants. The new nanovaccine formulation using partially-purified antigens along with nanoparticle adjuvants offers an alternative strategy for making cheaper veterinary vaccines.

Inspired by nature, synthetic mineralizing proteins have been developed to synthesize various structures of silica-based nanomaterials under environmentally friendly conditions. However, the development of bioprocesses able to assist in the translation of these new materials has lagged the development of the materials themselves. The development of cost-effective and scalable bioprocesses which minimize reliance on chromatography to recover biomolecules from microbial cell factories remains a significant challenge. This paper reports a simplified purification process for a recently reported recombinant catalytic modular (D4S2) protein (M(DPSMKQLADS-LHQLARQ-VSRLEHA)4 EPSRKKRKKRKKRKKGGGY; M 13.3 kDa; pI 10.9), which combines a variant of the established designer biosurfactant protein DAMP4 with a new biomimetic sequence (RKKRKKRKKRKKGGGY), providing for a bi-modular functionality (emulsification and biosilicification). The four-helix bundle structure of the protein has been demonstrated to remain stable and soluble under high temperature and high salt conditions, which confers simplified bioprocessing character. However, the high positive charge on the biosilification sequence necessitates removal of DNA contaminants from crude cell-extract at an early stage in the process by adding poly(ethyleneimine) (PEI). In this process, cellular protein contaminants were selectively precipitated by adding Na2 SO4 to the protein mixture up to a high concentration (1 M) and mixed at high temperature (90°C, 5 min) where D4S2 remained stable and soluble due to its four-helix bundle structure. Further increase of the Na2 SO4 concentration to 1.8 M precipitated, thus separated, D4S2 from residual PEI. The overall yield of the protein D4S2 was 28.8 mg per 800 mL cells (final cultivation OD600 ∼2) which gives an approximate 79% D4S2-protein yield. In comparison with the previously reported chromatographic purification of D4S2 protein (Wibowo et al., 2015), the final yield of D4S2 protein is increased fourfold in this study. The bio-produced protein D4S2 was proved to retain it emulsification and biosilicification functionalities enabling the formation of oil-core silica-shell nanocapsules at near-neutral pH and room temperature without the use of any toxic organic solvents, confirming no adverse effects due to bioprocess simplification. This work demonstrates that, through proper bioprocess engineering including the removal of critical contaminants such as DNA, a more efficient, simple, and scalable purification process can be used for the high-yield bio-production of a recombinant templating protein useful in the synthesis of bio-inspired nanomaterials. This simplified process is expected to be easily adapted to recover other mineralizing helix bundle-based functional proteins from microbial cell factories. Biotechnol. Bioeng. 2017;114: 335-343. © 2016 Wiley Periodicals, Inc.

We recently developed a novel approach for making oil-core silica-shell nanocapsules using designed bifunctional peptides (also called biomineralizing peptide surfactants) having both surface activity and biomineralization activity. Using the bifunctional peptides, oil-in-water nanoemulsion templates can be readily prepared, followed by the silicification directed exclusively onto the oil droplet surfaces and thus the formation of the silica shell. To explore their roles in the synthesis of silica nanocapsules, two bifunctional peptides, AM1 and SurSi, were systematically studied and compared. Peptide AM1, which was designed as a stimuli-responsive surfactant, demonstrated quick adsorption kinetics with a rapid decrease in the oil-water interfacial tension, thus resulting in the formation of nanoemulsions with a droplet size as small as 38 nm. Additionally, the nanoemulsions showed good stability over 4 weeks because of the formation of a histidine-Zn(2+) interfacial network. In comparison, the SurSi peptide that was designed by modularizing an AM1-like surface-active module with a highly cationic biosilicification-active module was unable to effectively reduce the oil-water interfacial tension because of its high molecular charge at neutral pH. The slow adsorption resulted in the formation of less stable nanoemulsions with a larger size (60 nm) than that of AM1. Besides, both AM1 and SurSi were found to be able to induce biomimetic silica formation. SurSi produced well-dispersed and uniform silica nanospheres in the bulk solution, whereas AM1 generated only irregular silica aggregates. Consequently, well-defined silica nanocapsules were synthesized using SurSi nanoemulsion templates, whereas silica aggregates instead of nanocapsules predominated when templating AM1 nanoemulsions. This finding indicated that the capability of peptide surfactants to form isolated silica nanospheres might play a role in the successful fabrication of silica nanocapsules. This fundamental study provides insights into the design of bifunctional peptides for making silica nanocapsules.

Quaternary ammonium containing compounds (QACs) such as cetylpyridinium chloride (CPC) is commonly employed in hyaluronic acid (HA) production process as an HA precipitating agent. 3(Trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride, a Si containing QAC (Si-QAC) generally used to modify the surface of cotton fibers for the preparation of nonleaching antibacterial textiles, has a chemical structure very similar to CPC. Choline, a natural QAC, can form a deep eutectic solvent with urea and be grafted onto cotton surface when incubating cotton in the deep eutectic solvent. Both of the QAC-modified cottons demonstrated antibacterial activity against Gram () Escherichia con and Gram (+) Bacillus subtilis along with HA adsorption capacity. The HA adsorption isotherm could be well-fitted with Langmuir model with maximum adsorption capacities of 184 mg/g and 351 mg/g for Si-QAC and choline surface-functionalized cotton fibers, respectively. In the presence of high concentration of contaminants, HA could be directly recovered from a B. subtilis culture with a capacity of 15 mg/g by using Si-QAC modified antimicrobial cotton fiber. (C) 2010 Elsevier B.V. All rights reserved.

The adsorption behavior of activated carbons is determined not only by their porous structures but also by the chemical nature of its surface. The surface chemistry of activated carbons can be selectively modified in order to improve their adsorption capacity. In this study, a NORIT granular activated carbon was treated by oxidant (HNO 3 ) and non-oxidant acid (HCI) at different concentrations and temperatures. The surface chemistries of the materials were characterized by Boehm titration method and by the determination of the point of zero charge (pH PZC ).The adsorption properties of the selected samples were studied by adsorption of methylene blue, which is one of the important dyes and found in many textile effluents. In addition, the pore structures of the modified carbons were also studied by argon adsorption at 87.29 K. As results, it was observed that both HN0 3 and HCI treatments could increase the surface acidity of activated carbons. Activated carbons modified by HCI gave the best performance on the adsorption of methylene blue. Keywords: Activated Carbon, Surface Chemistry, Chemical Treatment, Boehm Titration Method, Adsorption Abstrak Kemampuan adsorpsi karbon akti.ftidak hanya ditentukan oleh struktur pori tetapijuga dipengaruhi oleh sifat kimia dari permukaannya. Sifat kimia permukaan karbon aktif dapat secara selektif dimodifikasi dengan tujuan untuk lebih meningkatkan kapasitas adsorpsinya. Pada penelitian ini, karbon aktif NORIT granular ditreatment dengan menggunakan asam oksidator (HNO) dan non-oksidator (HCI) pada berbagai konsentrasi dan suhu. Sifat kimia permukaan karbon aktif dikarakterisasi dengan menggunakan metode titrasi Boehm serta dengan penentuan point of zero charge (pH PZC ). Kemampuan adsorpsinya diuji dengan mengadsorp larutan methylene blue, dimana methylene blue merupakan salah satu komponen dalam limbah tekstil. Sedangkan struktur pori karbon aktif dianalisa dengan adsorpsi Ar pada suhu 87,29 K. Penelitian ini menunjukkan bahwa baik treatment dengan HNO 3 maupun HCI dapat mengakibatkan terjadinya peningkatan sifat asam pada permukaan karbon aktif. Karbon aktif yang diberi perlalatan dengan HCI memberikan kemampuan adsorpsi yang paling baik dalam adsorpsi larutan methylen biru. Kata Kunci: Karbon Aktif, Sifat Kimia Permukaan, Perlakuan dengan Larutan Kimia, Metode Titrasi Boehm, Adsorpsi

The influence of surface chemistry and solution pH on the adsorption of benzene and toluene on activated carbon and its acid and heat treated forms were studied. A commercial coal-based activated carbon F-400 was chosen as carbon parent. The carbon samples were obtained by modification of F-400 by means of chemical treatment with HNO3 and thermal treatment under nitrogen flow. The treatment with nitric acid caused the introduction of a significant number of oxygenated acidic surface groups onto the carbon surface, while the heat treatment increases the basicity of carbon. The pore characteristics were not significantly changed after these modifications. The dispersive interactions are the most important factor in this adsorption process. Activated carbon with low oxygenated acidic surface groups (F-400Tox) has the best adsorption capacity.

Silica nanocapsules having corenshell architecture are of great interest in many current and emerging areas of technology. The core provides high-capacity loading of various actives, while the silica shell serves as protective envelope and diffusion barrier enabling controlled release of the active. Currently, production of silica nanocapsules is mainly based on templating methods using chemical surfactants to stabilize the template and to induce hydrolysis and condensation of silica precursors forming solid silica shell surrounding the template. Major drawbacks with the current methods are extreme pH conditions and/or elevated temperatures involving toxic chemicals that might pose undesirable environmental side effects and require tedious procedures for removing the chemicals. Inspired by Nature, biomimetic approaches have recently been developed to synthesize silica-based nanomaterials under mild physiological conditions through the use of biomolecules (i.e., peptides and proteins). This contrast in processing conditions and the growing demand for benign synthesis methods having minimum environmental risks have spurred much interest in biomimetic approaches. However, their utility is limited to biosilicification in bulk aqueous solution or at solidnliquid interfaces. There is no current biosilicification at oilnwater interfaces, and thus fabrication of oil-core silica-shell nanocapsules using biomolecules has not been realized yet. This thesis addresses the question on benign synthesis method for forming biocompatible oil-core silica-shell nanocapsules, by designing and utilizing biomolecules, in lieu of chemical surfactants, to facilitate formation and stabilization of oil droplets and to direct nucleation and growth of silica shells at the oilnwater interfaces. The major outcomes of the work presented in this thesis are: (1) development of a designed bifunctional modular peptide, SurSi, comprising a surface-active module and a biosilicification-active module that led to novel emulsion and biomimetic dual-templating approach for making oil-core silica-shell nanocapsules with tunable silica shell thickness; (2) facile encapsulation and sustained release of model insecticide, fipronil, from the silica nanocapsules in vitro in water and in vivo against a model insect, termite; (3) development of a designed bifunctional modular protein, D4S2, for making silica nanocapsules, thereby extending conceptual design of SurSi peptide, so that the protein could be produced renewably through biological expression in industrially-relevant, microbial cell-factory Escherichia coli (E. coli); and (4) large-scale production of D4S2 protein for synthesis of fipronil-encapsulated silica nanocapsules which could then be used for termite field trial. This work introduces, to the best of our knowledge, the first design and use of bifunctional modular biomolecules for making oil-core silica-shell nanocapsules. The dual-templating method developed in this thesis represents a new strategy for forming silica nanocapsules using components and processes expected to have minimal environment footprint. Active molecule can be easily encapsulated in silica nanocapsules with high encapsulation efficiency, by directly dissolving the active in the oil core prior to silica shell formation, and then released controllably from the nanocapsules with the release kinetics depending on the silica shell thicknesses. Furthermore, the use of bifunctional modular protein that can be produced through E. coli expression system has opened opportunities for sustainable and scalable bioprocess routes to produce oil-filled silica nanocapsules, thus makes the platform developed in this thesis suitable for large-scale applications especially in the fields of biomedical and agricultural applications that demand biocompatibility, high encapsulation efficiency and controlled-release properties.

A novel, bio-inspired templating platform technology is reported for the synthesis of biocompatible oil-core silica-shell nanocapsules with tunable shell thickness by utilizing a designed bifunctional peptide. Furthermore, facile encapsulation of an active molecule and its sustained release are demonstrated.