Protein Microspheres Research Articles

Protein Nanospheres and Nanofibers Prepared by Ice-Templating for the Controlled Release of Hydrophobic Drugs.

Protein scaffolds play a vital role in drug delivery systems. However, few research studies have been focused on loading hydrophobic drugs on protein scaffolds in biomedical fields. Here, we report on the development of protein microspheres and nanofibers by a simple ice-templating approach and their use as scaffolds for the controlled release of hydrophobic drugs, with bovine serum albumin (BSA) as the model protein and curcumin as the model hydrophobic drug. The BSA scaffolds display the unique nanofibrous and microspherical structures. This is a surprising discovery because there has been no report on the formation of microspheres via simple ice-templating of solutions or suspensions. To further understand the formation of microspheres by this approach, lysozyme, papain, and their composites with BSA are also studied. It is speculated that nanoparticles are first formed in aqueous BSA solution, attributed to the overlapping of hydration layers and autoassembly of inner hydrophobic cores of BSA globular molecules. Nanoprecipitation and soaking evaporation approaches are then used to load curcumin into the BSA scaffolds, followed by cross-linking with glutaraldehyde vapor to improve stability in an aqueous medium. The controlled release of curcumin is demonstrated, paving the way for various hydrophobic drugs loaded into this biodegradable and nonimmunogenic protein scaffold for potential treatments of diverse diseases.

Target-specific affinity separation of the bioactive compounds from herbal extract using the spin column packed with the immobilized protein microspheres prior to LC-MS analysis

Excellent pretreatments before instrumental analysis are critical for separation and determination of target compounds for discovery of new drugs from herb medicines. We developed a rapid and highly-selective method to separate the bioactive compounds from herbal extract using protein affinity-selection spin column, which was packed with the new sorbent materials from integrating the recombinant β2-adrenoceptor (β2-AR) directly out of cell lysates onto the surface of microspheres. Protein affinity-selection spin column was placed in a centrifugal tube, where after the non-specific binders were released to the filtrate under the operational centrifugation, the specific binders on the spin column were cleaned with a washing solvent for LC-MS analysis. The known agonists of β2-AR were retained/released on protein affinity-selection spin column but not on control column, demonstrating the method with good recovery (79.4∼95.7 %) and high repeatability (RSD < 3.5 %). The adsorption features of three ligands on the spin column were described best by Prism saturation binding model, and the high-affinity binding and the large binding capacity of the spin column make it feasible to trap the trace analytes effectively. It was applied in separating bioactive compounds from Alstoniae Scholaris extract, two of which were identified as picrinine and oleanolic acid in combination with LC-MS and verified as the potential agonists towards β2-AR though molecular docking and cell experiments. Our study demonstrated that, the spin column with the immobilized protein sorbents in the centrifugal filter device represents a promising tool, enabling rapid and target-specific affinity separation of the bioactive compounds from herbal extract.

Hybrid protein microspheres and their responsive release behaviors and inhibitory effects on melanin synthesis.

In this study, the formation of protein microspheres through lysosomal enzyme-assisted biomineralized crystallization was demonstrated. Spherical micro-sized hybrid CaCO3 constructs were synthesized and characterized using field-emission scanning electron microscopy equipped with energy-dispersive X-ray spectroscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, and particle size analysis. Additionally, parameters such as the Brunauer-Emmett-Teller surface area and single-point total pore volume, and adsorption/desorption analysis were used to investigate the mesoporous properties, which are advantageous for lysosomal enzyme (LE) loading. A LE can be used as an organic template, not only as a morphological controller but also for entrapping LE during the crystallization pathway. The hybrid protein microspheres accommodated 2.3 mg of LE with a 57% encapsulation efficiency and 5.1 wt% loading. The peroxidase activity of the microspheres was calculated and found to be approximately 0.0238 mM-1 min-1. pH-responsive release of the LE from CaCO3 was observed, suggesting potential biomedical and cosmetic applications in acidic environments. The hybrid LE microsphere treatment significantly alleviated melanin production in a dose-dependent manner and further downregulated the mRNA expression of MITF, tyrosinase, TYRP-1, and TYRP-2. These results indicate skin-whitening effects by inhibiting melanin without inducing cytotoxicity. The data provide the first evidence of the potential use of a LE for obtaining hybrid minerals and the effectiveness of biomineralization-based sustainable delivery of enzyme-based vehicles based on organelle-extract-assisted biomineralization.

Electrochemical Active Micro‐Protein Coating by Self‐Assembling 2D‐Microfluidics for Stabilizing Lithium Metal Anode

AbstractMicrofluidics is of great interest for nano‐/micro‐fabrication but is conventionally limited inside microchannels. Breaking this restriction and developing 2D‐microfluidics is anticipated to expand the potential of microfluidics. Here, by harnessing the capillary effect of porous battery separator, a concept of self‐assembling capillary 2D‐microfluidics is proposed to achieve well‐controlled functional coating onto the separator thin‐film. The capillary‐assisted liquid tailoring behavior of this 2D‐microfluidics is investigated by both experimental and simulation studies, and the capillary number is found as the key parameter controlling the 2D‐micofluid thickness. For application studies, zein protein solution is employed for this 2D‐microfluidics to generate electrochemical active and self‐assembled protein microsphere coating onto the separator after drying. The resultant protein microsphere functionalized separator (PMFS) can physiochemically stabilize the surface of lithium metal anode. First, the PMFS works as spherical template to regulate uniform deposition of lithium ions. Second, similar to sustained drug release, the PMFS releases dissolved protein as functional additive into liquid electrolyte, assisting to form robust solid‐electrolyte‐interphase with highly conductive Li–C–N component. This work not only proposes a facile self‐assembling 2D‐microfluidic technology for surface nano‐/micro‐fabrication, but also brings forth a promising protein‐based physicochemical strategy for stabilizing lithium metal anode.

Water-in-Oil Pickering Emulsions Stabilized by Hydrophobized Protein Microspheres.

Water-in-oil (w/o) Pickering emulsions have gained considerable attention in colloid science and daily applications. However, for the formation of w/o emulsions, especially those with high internal water content, the particulate stabilizers are required to be sufficiently hydrophobic, and synthetic or chemically modified particles have been mostly reported until now, which are not biocompatible and sustainable. We present a zein protein-based microsphere derived from the Pickering emulsion template, in which protein microspheres are feasibly in situ hydrophobized by silica nanoparticles, enabling the stabilization of w/o Pickering emulsions. The effects of microsphere concentration, water/oil volume ratio, oil types, and pH on the stabilization of prepared w/o emulsions are systematically studied, revealing prominent characteristics of the controllable size, high water fraction, universal adaptation of oils, as well as broad pH stability. As a demonstration, the Pickering emulsion effectively encapsulates vitamin C and shows high stability for long storage duration against ultraviolet radiation/heat. Therefore, this novel proteinaceous particle-stabilized w/o Pickering emulsion has great potential in the delivery and protection of water-soluble bioactive substrates.

One-step preparation of macroporous zein microspheres by solvent diffusion for dye adsorption

Macroporous microspheres have attracted significant interest in various fields since macropores endow with fast mass transfer. However, constructing the macropore mostly requires additional porogenic agents and complex steps. Here, a simple solvent diffusion strategy has been developed for the rapid preparation of macroporous protein microspheres. Based on the strategy, the macropores in the microspheres are created by the removal of the trapped aqueous phase. The results show that the average particle size of the prepared microspheres is centered at about 10 µm. The pore size of the microspheres ranges from 50 nm to about 1 µm and can be controlled by adjusting the parameters of surfactant, temperature, and concentration during the preparation process for different requirements. Finally, the macropore microspheres are characterized by the adsorption of Congo red dyes, which show excellent adsorption ability. According to these results, these macroporous protein microspheres have a practical potential for applications in adsorption and biomedicine fields.

Biological miniature temperature sensor based on monodisperse microsphere lasers fabricated by soft microfluidic technology

Microsphere biolasers employing biological materials as their cavity matrix have attracted tremendous research attention due to their potential for bio-integration, cell-tracking and biosensing. Several techniques have been applied for fabricating microsphere biolasers such as emulsion and vacuum freeze-drying but the current technology generally can not control the output size of the laser and therefore hinder them from many applications. In this work, we demonstrate that a low-cost microfluidic device can be very effective in fabricating nearly monodisperse dye-doped protein microspheres with up to 70% of them having the same size. Under optical pumping, these microspheres emit lasing emission with a lasing threshold of ∼1 µJ and a quality factor of ∼2.5 × 103. The lasing mechanism is ascribed to whispering gallery mode. Furthermore, the obtained microlasers can be employed for temperature sensing based on the wavelength shift of lasing mode with increasing temperature. The sensor sensitivity in the measured range of 25 °C–50 °C is about 0.47 nm/°C. More interestingly, microlasers of the same size exhibit a very similar sensing performance which confirm their high reproducibility and reliability. Owing to the biocompatibility and small size, these miniature laser-based sensors can be implantable in skins and tissues for biological studies and medical diagnostic.

Biocompatible Polymer and Protein Microspheres with Inverse Photonic Glass Structure for Random Micro‐Biolasers

Random Micro-Biolasers Micro-sized random lasers made with organic materials are interesting for many photonic applications, but their fabrication is challenging. In article number 2100036, Van Duong Ta, Riccardo Sapienza, and co-workers demonstrate a versatile technique for fabricating micro-scale random lasers with diameters ranging from 30 to 160 μm. The lasers are synthesized from dye-doped polyvinyl alcohol or bovine serum albumin and have a microporous structure with pores of 1.28 μm.

Biocompatible Polymer and Protein Microspheres with Inverse Photonic Glass Structure for Random Micro‐Biolasers

The miniaturization of random lasers to the micrometer scale is challenging but fundamental for the integration of lasers with photonic integrated circuits and biological tissues. Herein, it is demonstrated that random lasers with a diameter from 30 to 160 μm can be achieved by using a simple emulsion process and selective chemical etching. These tiny random laser sources are made of either dye‐doped polyvinyl alcohol (PVA) or bovine serum albumin (BSA) and they are in the form of microporous spheres with monodisperse pores of 1.28 μm in diameter. Clear lasing action is observed when the microporous spheres are optically excited with powers larger than the lasing threshold, which is 154 μJ mm−2 for a 75 μm diameter PVA microporous sphere. The lasing wavelength redshifts 10 nm when the PVA microsphere diameter increases from 34 to 160 μm. For BSA microspheres, the lasing threshold is around 55 μJ mm−2 for a 70 μm diameter sphere and 104 μJ mm−2 for a 35 μm diameter sphere. The simple fabrication process reported allows for detail studies of morphology and size, important for fundamental studies of light–matter interaction in complex media, and applications in photonic integrated circuits, photonic barcoding, and optical biosensing.

Monodisperse and size-tunable high-quality factor microsphere biolasers.

Microsphere biolasers have attracted a great deal of interest due to their potential for biosensing and cell tracking. Here we demonstrate a novel, to the best of our knowledge, microfluidic-based fabrication of nearly monodisperse dye-doped protein microsphere biolasers with a tunable size from 150 to 50 µm. In particular, for an 85 µm-bead, about 70% of the fabricated microspheres have the same size of 85 µm. Under optical pumping, the fabricated microspheres emit whispering gallery mode lasing emission with a lasing threshold of ${{7}}\;\unicode{x00B5} {\rm{J}}\;{{\rm{mm}}^{- 2}}$ and quality ($\!Q$) factor up to 3000. Interestingly, microspheres with the same size exhibit a similar lasing threshold and spectrum. The result indicates a high reproducibility of microsphere biolasers by the microfluidic-based fabrication technique. This Letter provides an effective method for mass production of high-$Q$ factor microsphere biolasers which is a significant step toward real biosensing and medical applications.

Preparation of silk fibroin-based microspheres under high-gravity field

The possibility of preparing regenerated silk fibroin (RSF)-based microspheres by a high-gravity anti-solvent precipitation method was explored. The high-gravity environment was generated by a rotating packed bed (RPB) reactor. Aqueous RSF solutions and ethanol were used as solvent and anti-solvent, respectively. The effects of liquid flow rates and anti-solvent/solvent ratios, as well as centrifugation and freezing time, on the formation, size distribution and morphology of RSF microspheres were investigated. The results showed that RSF microspheres, with diameters of 0.1–1.6 μm, can be prepared under high-gravity field via self-assembly of RSF molecular chains induced by ethanol and quenched by freezing and lyophilization. The mechanism of RSF microsphere formation was also discussed. In addition, rhodamine B was used as a model drug for evaluation of drug delivery capability of the RSF microspheres. The merits of this study lie in a simple low-cost apparatus with ease for scale-up and thereby potential high volume continuous production capacity, which is of practical advantages and industrial significance in preparation of protein microspheres for commercial mass production.

Protein microspheres as structuring agents in lipids: potential for reduction of total and saturated fat in food products.

Excess consumption of total and saturated fats is linked to the development of chronic diseases, such as obesity, heart disease, diabetes, and cancer. There is therefore considerable interest in the development of foods containing lower levels of total and saturated fats, but that still have the same desirable physicochemical and sensory characteristics as the original foods. Solid fats normally contribute a number of key functional attributes to foods due to their ability to form crystalline networks that alter texture (such as elasticity, plasticity, and spreadability) and appearance (such as opacity and creaminess). The aim of this review is to provide an overview and to discuss the potential applications of food proteins as fat structuring agents that may be able to offer some of the desirable attributes normally supplied by saturated and trans fats. Previous studies have shown that globular proteins (such as whey proteins) trapped inside water-in-oil emulsions form protein microspheres when they are thermally denatured, which leads to the creation of highly viscous or solid-like lipid phases, having higher rheological properties. These protein microspheres may therefore be useful for the development of reduced fat margarines and spreads with reduced level of saturated/trans-fat contents. © 2020 Society of Chemical Industry.

Preparation and characterization of soy protein microspheres using amorphous calcium carbonate cores

Porous calcium carbonate (CaCO3) vaterite microparticles have been introduced a decade ago as sacrificial cores and becoming nowadays as one of the most popular templates to fabricate microcarriers. Herein we present a fabrication method of soy protein microspheres by templating on porous CaCO3 templates. The CaCO3 templates with a particle size (appx. 3–12 μm) were harvested with a yield of 47%. Soy proteins at varied concentrations from 0.05 to 0.25% (w/v) were applied to fabricate microspheres. Results showed that the highest coating efficiency of 87.9% was achieved at the protein concentration of 0.05%. And the highest protein loading ratio is 55.5 μg/mg. The protein coated CaCO3 microparticles has a spherical shape, however soy protein microspheres formed by dissolving the carbonate templates were wrinkled and partially collapsed. Results further indicated that the secondary structure of microspheres became more ordered with enhanced thermal stability. This study illustrates the feasibility to apply soy protein in the fabrication of microspheres based on calcium carbonate microparticles.

Cross-protective immune responses against African horse sickness virus after vaccination with protein NS1 delivered by avian reovirus muNS microspheres and modified vaccinia virus Ankara

African horse sickness virus (AHSV) is an insect-borne pathogen that causes acute disease in horses and other equids. In an effort to improve the safety of currently available vaccines and to acquire new knowledge about the determinants of AHSV immunogenicity, new generation vaccines are being developed. In this work we have generated and tested a novel immunization approach comprised of nonstructural protein 1 (NS1) of AHSV serotype 4 (AHSV-4) incorporated into avian reovirus muNS protein microspheres (MS-NS1) and/or expressed using recombinant modified vaccinia virus Ankara vector (MVA-NS1). The protection conferred against AHSV by a homologous MS-NS1 or heterologous MS-NS1 and MVA-NS1 prime/boost was evaluated in IFNAR (−/−) mice. Our results indicate that immunization based on MS-NS1 and MVA-NS1 afforded complete protection against the infection with homologous AHSV-4. Moreover, priming with MS-NS1 and boost vaccination with MVA-NS1 (MS-MVA-NS1) triggered NS1 specific cytotoxic CD8 + T cells and prevented AHSV disease in IFNAR (−/−) mice after challenge with heterologous serotype AHSV-9. Cross-protective immune responses are highly important since AHS can be caused by nine different serotypes, which means that a universal polyvalent vaccination would need to induce protective immunity against all serotypes.

IC-Tagging methodology applied to the expression of viral glycoproteins and the difficult-to-express membrane-bound IGRP autoantigen

We have previously developed a methodology to produce protein microspheres (MS) that can be loaded with proteins of interest in living cells through their C or N-terminal tagging with the so-called IC-Tag. The IC-Tagging method has many applications ranging from the production of immobilized enzymes for industrial use to the production of subunit vaccines due to its intrinsic adjuvancy. Here we show the adaptation of the IC-Tagging to work inside the endoplasmic reticulum and bacteria, allowing us to produce properly modified viral glycoproteins. Additionally, we were able to express the Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), whose expression remained elusive to date possibly due to its toxicity when over-expressed. IGRP is an antigen of enormous pharmaceutical interest as it is specifically targeted during the autoimmune response taking place in both the Non-Obese Diabetic (NOD) mice and type 1 diabetes (T1D) patients leading to the destruction of insulin-producing beta cells.

Characterization of Magnetic Nanoparticle-Seeded Microspheres for Magnetomotive and Multimodal Imaging.

Magnetic iron-oxide nanoparticles have been developed as contrast agents in magnetic resonance imaging (MRI) and as therapeutic agents in magnetic hyperthermia. They have also recently been demonstrated as contrast and elastography agents in magnetomotive optical coherence tomography and elastography (MM-OCT and MM-OCE, respectively). Protein-shell microspheres containing suspensions of these magnetic nanoparticles in lipid cores, and with functionalized outer shells for specific targeting, have also been demonstrated as efficient contrast agents for imaging modalities such as MM-OCT and MRI, and can be easily modified for other modalities such as ultrasound, fluorescence, and luminescence imaging. By leveraging the benefits of these various imaging modalities with the use of only a single agent, a magnetic microsphere, it becomes possible to use a widefield imaging method (such as MRI or small animal fluorescence imaging) to initially locate the agent, and then use MM-OCT to obtain dynamic contrast images with cellular level morphological resolution. In addition to multimodal contrast-enhanced imaging, these microspheres could serve as drug carriers for targeted delivery under image guidance. Although the preparation and surface modifications of protein microspheres containing iron oxide nanoparticles has been previously described and feasibility studies conducted, many questions regarding their production and properties remain. Since the use of multifunctional microspheres could have high clinical relevance, here we report a detailed characterization of their properties and behavior in different environments to highlight their versatility. The work presented here is an effort for the development and optimization of nanoparticle-based microspheres as multi-modal contrast agents that can bridge imaging modalities on different size scales, especially for their use in MM-OCT and MRI imaging.

Novel Iron-Whey Protein Microspheres Protect Gut Epithelial Cells from Iron-Related Oxidative Stress and Damage and Improve Iron Absorption in Fasting Adults

Background: Iron food fortification and oral iron formulations are frequently limited by poor absorption, resulting in the widespread use of high-dose oral iron, which is poorly tolerated. Methods: We evaluated novel iron-denatured whey protein (Iron-WP) microspheres on reactive oxygen species (ROS) and viability in gut epithelial (HT29) cells. We compared iron absorption from Iron-WP versus equimolar-dose (25 mg elemental iron) ferrous sulphate (FeSO₄) in a prospective, randomised, cross-over study in fasting volunteers (n = 21 per group) dependent on relative iron depletion (a ferritin level ≤/>30 ng/mL). Results: Iron-WP caused less ROS generation and better HT29 cell viability than equimolar FeSO₄. Iron-WP also showed better absorption with a maximal 149 ± 39% increase in serum iron compared to 65 ± 14% for FeSO₄ (p = 0.01). The response to both treatments was dependent on relative iron depletion, and multi-variable analysis showed that better absorption with Iron-WP was independent of baseline serum iron, ferritin, transferrin saturation, and haemoglobin in the overall group and in the sub-cohort with relative iron depletion at baseline (p < 0.01). Conclusions: Novel Iron-WP microspheres may protect gut epithelial cells and improve the absorption of iron versus FeSO₄. Further evaluation of this approach to food fortification and supplementation with iron is warranted.

Collagen I derived recombinant protein microspheres as novel delivery vehicles for bone morphogenetic protein-2

Bone morphogenetic protein-2 (BMP-2) is a powerful osteoinductive protein; however, there is a need for the development of a safe and efficient BMP-2 release system for bone regeneration therapies. Recombinant extracellular matrix proteins are promising next generation biomaterials since the proteins are well-defined, reproducible and can be tailored for specific applications. In this study, we have developed a novel and versatile BMP-2 delivery system using microspheres from a recombinant protein based on human collagen I (RCP). In general, a two-phase release pattern was observed while the majority of BMP-2 was retained in the microspheres for at least two weeks. Among different parameters studied, the crosslinking and the size of the RCP microspheres changed the in vitro BMP-2 release kinetics significantly. Increasing the chemical crosslinking (hexamethylene diisocyanide) degree decreased the amount of initial burst release (24h) from 23% to 17%. Crosslinking by dehydrothermal treatment further decreased the burst release to 11%. Interestingly, the 50 and 72μm-sized spheres showed a significant decrease in the burst release compared to 207-μm sized spheres. Very importantly, using a reporter cell line, the released BMP-2 was shown to be bioactive. SPR data showed that N-terminal sequence of BMP-2 was important for the binding and retention of BMP-2 and suggested the presence of a specific binding epitope on RCP (KD: 1.2nM). This study demonstrated that the presented RCP microspheres are promising versatile BMP-2 delivery vehicles.

Injectable silk fibroin hydrogels functionalized with microspheres as adult stem cells-carrier systems

Hydrogels are good candidate materials for cell delivery scaffolds because they can mimic the physical, chemical, electrical and biological properties of most of the native tissues. In this study, composite biosynthetic hydrogels were produced by combining the bio-functionality of silk fibroin (SF) with the structural versatility of polyethylene-glycol-diacrylated (PEGDa). The formation of a photopolymerizable PEGDa-SF hydrogel (PSFHy) was optimized for 3D-cell culture. Functionalization of the 3D-PSFHy with protein microspheres (MS) was required to increase the porosity and cell-adhesive properties of the material. Cardiac mesenchymal stem cells, which were cultured within the MS-embedding PSFHy, exhibited good viability and expression of proteins that are characteristic of the initial phases of the cardiac muscle differentiation process. Further, the addition of chondroitin sulfate into the scaffolds improved the cell viability. A cell-preconditioning of the scaffold was also performed, suggesting a potential application of these sponge-like scaffolds for analysing the effects of several extracellular microenvironments, produced by different kinds of cells, on the stem cells fate. The results presented herein highlight on the possibility to use the PSFHys functionalized with MS as stem cell-carrier systems with sponge-like properties, potential ultrasound-imaging contrast agents and controlled biochemical factor delivery.

A New Approach to Chiral Enrichment by Exposure of Racemates of Amino Acids to Sonochemically‐Prepared BSA Microspheres

AbstractThis preliminary study describes a one‐step sonochemical formation of Bovine Serum Albumin (BSA) microspheres and their chiral interaction with several amino acids. Partial separation between enantiomers of various amino acids was accomplished by the interaction of these microspheres with solutions of racemic amino acids. The highest rate of enantiomeric enrichment was attained for D,L‐cysteine, where 59% enantiomeric excess (ee) of the L‐cysteine was measured. This indicates the preferrential interaction of the BSA microspheres with the D‐enantiomer of cystein. No chiral activity was observed for unsonicated BSA molecules. All other D,L‐amino acids that were examined provided enrichment of the D‐enantiomer albeit in lower ee values. D,L‐cysteine interacted with the BSA microspheres leading to L‐enantioselective enrichment up to 59 % ee at room tempertaure and 72% at 5 °C. Chiral enrichment was also carried out succesfully with other protein microspheres such as Avidin microspheres. In this case up to 41 % ee was acheived on interaction with DL‐phenylalanine. Our simple and facile method opens a new window for enantiomeric separation of racemic compounds.