David Wibowo

Sustained release of fipronil insecticide in vitro and in vivo from biocompatible silica nanocapsules.

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.

Interfacial biomimetic synthesis of silica nanocapsules using a recombinant catalytic modular protein

This paper reports interfacially driven synthesis of oil-core silica-shell nanocapsules using a rationally designed recombinant catalytic modular protein (ReCaMoP), in lieu of a conventional chemical surfactant. A 116-residue protein, D4S2, was designed by modularizing a surface-active protein module having four-helix bundle structure in bulk and a biosilicification-active peptide module rich in cationic residues. This modular combination design allowed the protein to be produced via the industrially relevant cell factory Escherichia coli with simplified purification conferred by thermostability engineered in design. Dynamic interfacial tension and thin film pressure balance were used to gain an overview of the protein behavior at macroscopic interfaces. Functionalities of D4S2 to make silica nanocapsules were demonstrated by facilitating formation and stabilization of pharmaceutically grade oil droplets through its surface-active module and then by directing nucleation and growth of a silica shell at the oil-water interface through its biosilicification-active module. Through these synergistic activities in D4S2, silica nanocapsules could be formed at near-neutral pH and ambient temperature without using any organic solvents that might have negative environmental and sustainability impacts. This work introduces parallelization of biomolecular, scale-up and interfacial catalytic design strategies for the ultimate development of sustainable and scalable production of a recombinant modular protein that is able to catalyze synthesis of oil-filled silica nanocapsules under environmentally friendly conditions, suitable for use as controlled-release nanocarriers of various actives in biomedical and agricultural applications.

Emulsion-templated silica nanocapsules formed using bio-inspired silicification

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.

Interfacial engineering for silica nanocapsules.

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.

Non‐chromatographic bioprocess engineering of a recombinant mineralizing protein for the synthesis of silica nanocapsules

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)4EPSRKKRKKRKKRKKGGGY; 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 Na2SO4 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 Na2SO4 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.

Insights into the role of biomineralizing peptide surfactants on making nanoemulsion-templated silica nanocapsules

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.

Microfluidic self-assembly of a combinatorial library of single- and dual-ligand liposomes for in vitro and in vivo tumor targeting

ABSTRACT 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 72h) 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.

Understanding the Effects of Nanocapsular Mechanical Property on Passive and Active Tumor Targeting

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 Youngs 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.

Design of Modular Peptide Surfactants and Their Surface Activity

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 within 400 s, respectively. Their relatively quick adsorption kinetics allowed the formation of nanoemulsions with smaller droplet sizes and narrower size distribution. AM-S and C8-AM at 800 μM bulk concentration could make nanoemulsions of average diameters 180 and 147 nm, respectively, by simple sonication. With respect to the long-term stability, a minimum peptide concentration of 400 μM for AM-S and a lower concentration of 100 μM for C8-AM were demonstrated to effectively stabilize nanoemulsions over 3 weeks. Compared to AM1, the AM-S nanoemulsion retained its stimuli-responsive function triggered by metal ions, whereas the C8-AM nanoemulsions did not respond to the stimuli as efficiently as AM-S because of the strong anchoring ability of the hydrophobic C8 module. The two-module design of AM-S and C8-AM represents a new strategy in tuning the surface activity of peptide surfactants, offering useful information and guidance of future designs.

Synergetic Combinations of Dual‐Targeting Ligands for Enhanced In Vitro and In Vivo Tumor Targeting

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.