Nanoengineered Silica-Based Biomaterials for Regenerative Medicine.

The paradigm of regenerative medicine is undergoing a transformative shift with the emergence of nanoengineered silica-based biomaterials. Their unique confluence of biocompatibility, precisely tunable porosity, and the ability to modulate cellular behavior at the molecular level makes them highly desirable for diverse tissue repair and regeneration applications. Advancements in nanoengineered silica synthesis and functionalization techniques have yielded a new generation of versatile biomaterials with tailored functionalities for targeted drug delivery, biomimetic scaffolds, and integration with stem cell therapy. These functionalities hold the potential to optimize therapeutic efficacy, promote enhanced regeneration, and modulate stem cell behavior for improved regenerative outcomes. Furthermore, the unique properties of silica facilitate non-invasive diagnostics and treatment monitoring through advanced biomedical imaging techniques, enabling a more holistic approach to regenerative medicine. This review comprehensively examines the utilization of nanoengineered silica biomaterials for diverse applications in regenerative medicine. By critically appraising the fabrication and design strategies that govern engineered silica biomaterials, this review underscores their groundbreaking potential to bridge the gap between the vision of regenerative medicine and clinical reality.

Biominerals and Bioinspired Materials in Biosensing: Recent Advancements and Applications.

Inspired by nature's remarkable ability to form intricate minerals, researchers have unlocked transformative strategies for creating next-generation biosensors with exceptional sensitivity, selectivity, and biocompatibility. By mimicking how organisms orchestrate mineral growth, biomimetic and bioinspired materials are significantly impacting biosensor design. Engineered bioinspired materials offer distinct advantages over their natural counterparts, boasting superior tunability, precise controllability, and the ability to integrate specific functionalities for enhanced sensing capabilities. This remarkable versatility enables the construction of various biosensing platforms, including optical sensors, electrochemical sensors, magnetic biosensors, and nucleic acid detection platforms, for diverse applications. Additionally, bioinspired materials facilitate the development of smartphone-assisted biosensing platforms, offering user-friendly and portable diagnostic tools for point-of-care applications. This review comprehensively explores the utilization of naturally occurring and engineered biominerals and materials for diverse biosensing applications. We highlight the fabrication and design strategies that tailor their functionalities to address specific biosensing needs. This in-depth exploration underscores the transformative potential of biominerals and materials in revolutionizing biosensing, paving the way for advancements in healthcare, environmental monitoring, and other critical fields.

Biomimetic Materials for Skin Tissue Regeneration and Electronic Skin.

Biomimetic materials have become a promising alternative in the field of tissue engineering and regenerative medicine to address critical challenges in wound healing and skin regeneration. Skin-mimetic materials have enormous potential to improve wound healing outcomes and enable innovative diagnostic and sensor applications. Human skin, with its complex structure and diverse functions, serves as an excellent model for designing biomaterials. Creating effective wound coverings requires mimicking the unique extracellular matrix composition, mechanical properties, and biochemical cues. Additionally, integrating electronic functionality into these materials presents exciting possibilities for real-time monitoring, diagnostics, and personalized healthcare. This review examines biomimetic skin materials and their role in regenerative wound healing, as well as their integration with electronic skin technologies. It discusses recent advances, challenges, and future directions in this rapidly evolving field.

Tailored Functionalized Protein Nanocarriers for Cancer Therapy: Recent Developments and Prospects.

Recently, the potential use of nanoparticles for the targeted delivery of therapeutic and diagnostic agents has garnered increased interest. Several nanoparticle drug delivery systems have been developed for cancer treatment. Typically, protein-based nanocarriers offer several advantages, including biodegradability and biocompatibility. Using genetic engineering or chemical conjugation approaches, well-known naturally occurring protein nanoparticles can be further prepared, engineered, and functionalized in their self-assembly to meet the demands of clinical production efficiency. Accordingly, promising protein nanoparticles have been developed with outstanding tumor-targeting capabilities, ultimately overcoming multidrug resistance issues, in vivo delivery barriers, and mimicking the tumor microenvironment. Bioinspired by natural nanoparticles, advanced computational techniques have been harnessed for the programmable design of highly homogenous protein nanoparticles, which could open new routes for the rational design of vaccines and drug formulations. The current review aims to present several significant advancements made in protein nanoparticle technology, and their use in cancer therapy. Additionally, tailored construction methods and therapeutic applications of engineered protein-based nanoparticles are discussed.

Cordyceps mushroom with increased cordycepin content by the cultivation on edible insects.

Cordycepin is the major constituent of Cordyceps mushroom (or Cordyceps militaris) with therapeutic potential. Insects are the direct sources of nutrients for Cordyceps in nature. Therefore, optimized condition of Cordyceps cultivation for efficient cordycepin production was explored using six edible insects as substrates. The highest yield of cordycepin was produced by the cultivation on Allomyrina dichotoma and was 34 times that on Bombyx mori pupae. Among insect components, fat content was found to be important for cordycepin production. Especially, a positive correlation was deduced between oleic acid content and cordycepin production. The transcriptional levels of cns1 and cns2, genes involved in cordycepin biosynthesis, were higher in Cordyceps grown on A. dichotoma than on other insects tested. The addition of oleic acid to the substrates increased cordycepin production together with the transcriptional levels of cns1 and cns2. Therefore, Cordyceps with high content of cordycepin can be secured by the cultivation on insects.

Oriented multivalent silaffin-affinity immobilization of recombinant lipase on diatom surface: Reliable loading and high performance of biocatalyst.

Microbial lipases are widely used biocatalysts; however, their functional surface immobilization should be designed for successful industrial applications. One of the unmet challenges is to develop a practical surface immobilization to achieve both high stability and activity of lipases upon the large loading. Herein, we present a silaffin-based multivalent design as a simple and oriented approach for Bacillus subtilis lipase A (LipA) immobilization on economic diatom biosilica matrix to yield highly-stable activity with reliable loading. Specifically, silaffin peptides Sil3H, Sil3K, and Sil3R, as monovalent or divalent genetic fusion tags, selectively immobilized LipA on biosilica surfaces. Sil3K peptide fusion to LipA termini most efficiently produced high catalytic activity upon immobilization. The activity was 70-fold greater than that of immobilized wild-type LipA. Compared to single fusion, the double Sil3K fusion displayed 1.7 higher enzymatic loading combined with high catalytic performances of LipA on biosilica surfaces. The multivalent immobilized LipA was distributed uniformly on biosilica surfaces. The biocatalyst was stable over a wide pH range with 98% retention activity after 10 reuses. The stabilized lipase fusion was compatible with laundry detergents, making it an attractive biocatalyst for detergent formulations. These findings demonstrate that multivalent surface immobilization is a plausible method for developing high-performance biocatalysts suitable for industrial biotechnological applications.

Biomimetic and bioinspired silicifications: Recent advances for biomaterial design and applications.

The rational design and controllable synthesis of functional silica-based materials have gained increased interest in a variety of biomedical and biotechnological applications due to their unique properties. The current review shows that marine organisms, such as siliceous sponges and diatoms, could be the inspiration for the fabrication of advanced biohybrid materials. Several biomolecules were involved in the molecular mechanism of biosilicification in vivo. Mimicking their behavior, functional silica-based biomaterials have been generated via biomimetic and bioinspired silicification in vitro. Additionally, several advanced technologies were developed for in vitro and in vivo immobilization of biomolecules with potential applications in biocatalysis, biosensors, bioimaging, and immunoassays. A thin silica layer could coat a single living cell or virus as a protective shell offering new opportunities in biotechnology and nanomedicine fields. Promising nanotechnologies have been developed for drug encapsulation and delivery in a targeted and controlled manner, in particular for poorly soluble hydrophobic drugs. Moreover, biomimetic silica, as a morphogenetically active biocompatible material, has been utilized in the field of bone regeneration and in the development of biomedical implantable devices. STATEMENT OF SIGNIFICANCE: In nature, silica-based biomaterials, such as diatom frustules and sponge spicules, with high mechanical and physical properties were created under biocompatible conditions. The fundamental knowledge underlying the molecular mechanisms of biosilica formation could inspire engineers and chemists to design novel hybrid biomaterials using molecular biomimetic strategies. The production of such biohybrid materials brings the biosilicification field closer to practical applications. This review starts with the biosilicification process of sponges and diatoms with recently updated researches. Then, this article covers recent advances in the design of silica-based biomaterials and their potential applications in the fields of biotechnology and nanomedicine, highlighting several promising technologies for encapsulation of functional proteins and living cells, drug delivery and the preparation of scaffolds for bone regeneration.

Silaffin-3-derived pentalysine cluster as a new fusion tag for one-step immobilization and purification of recombinant Bacillus subtilis catalase on bare silica particles.

Bio-catalysis by enzymes on solid surfaces has been implemented in several practical applications. However, the current methods for efficient enzyme immobilization with retained activity need further development. Herein, a simple, rapid, and economical, bio-affinity-based approach was developed for the direct immobilization with high activity recovery of the Bacillus subtilis catalase (CAT), recombinantly expressed in Escherichia coli. Silaffin-3-derived pentalysine cluster (Sil3K) from Thalassiosira pseudonana and its mutant variant (penta-arginine peptide; Sil3R) were used for the first time in the non-covalent immobilization of the recombinant enzyme on silica particles. The fusion proteins CAT-Sil3K and CAT-Sil3R were selectively loaded from the cell lysates onto the silica surface. Unexpectedly, the Lys-based tag (Sil3K) was the superior to Arg-based tag (Sil3R) or tag-less system for the high recovery of CAT activity upon immobilization; an 8.4-fold and 1.5-fold increase in the catalytic activity was observed for CAT-Sil3K compared with the tag-less CAT and CAT-Sil3R, respectively. Furthermore, the CAT-Sil3K immobilized on silica particles exhibited improved thermal, pH and storage stabilities, and retained 72% of the initial activity after five reaction cycles. Moreover, CAT-Sil3K was released with approximately 85% recovery and 91% purity, in a biologically active form when free lysine solution was used as the eluent. Our data proved that Sil3K-tag, 12-mer peptide, can be a highly promising silica-affinity tag for effective enzyme immobilization with preserved activity. Additionally, the novel findings obtained here may open a new route not only for cost-effective enzyme immobilization approaches but also for high recovery of enzyme activity.

Direct immobilization and recovery of recombinant proteins from cell lysates by using EctP1-peptide as a short fusion tag for silica and titania supports.

Immobilization of protein, compared to the use of free protein, offers improved stability, easy separation and continuous reusability. However, the classic routes for protein immobilization, based on non-specific adsorption, often negatively affect protein functionality. In this study, EctP1 peptide was explored as a novel short fusion tag for non-covalent adsorption on unmodified solid surfaces, silica and titania. A fusion of EctP1 with bovine carbonic anhydrase (BCA) was employed to investigate the optimal binding conditions that could diminish the nonspecific adsorption of Escherichia coli proteins. The stable binding of BCA-EctP1 on titania was observed in the pH range of 2-9, while the stable binding on silica was in the pH range 6-9. Moreover, the immobilized BCA-EctP1 on silica and titania particles showed enhanced thermal and storage stability and retained 95% of its residual activity after 5 uses. We further demonstrated the merits of the noncovalent immobilization of EctP1 fusion proteins to silica and titania in the recovery of the bound proteins. Interestingly, monomeric arginine showed better recovery yield of EctP1 fusion proteins (about 78-84%), compared to the recovery yield by the salts, NaCl and MgCl2 (about 30-51%). Using BCA and monomeric red fluorescent protein (mRFP) as model proteins, the EctP1 fusion proteins were released in a biologically active form with approximately 80% recovery and 93% purity. Our approach is a simple and reproducible technique for direct immobilization of recombinant proteins from E. coli lysates on solid supports, with the potential high-purity recovery of recombinant proteins.

Anticancer effect of nor-wogonin (5, 7, 8-trihydroxyflavone) on human triple-negative breast cancer cells via downregulation of TAK1, NF-κB, and STAT3.

BACKGROUND: Nor-wogonin, a polyhydroxy flavone, has been shown to possess antitumor activity. However, the mechanisms responsible for its antitumor activity are poorly studied. Herein, we investigated the mechanisms of nor-wogonin actions in triple-negative breast cancer (TNBC) cells. METHODS: Effects of nor-wogonin on cell proliferation and viability of four TNBC cell lines (MDA-MB-231, BT-549, HCC70, and HCC1806) and two non-tumorigenic breast cell lines (MCF-10A and AG11132) were assessed by BrdU incorporation assays and trypan blue dye exclusion tests. Cell cycle and apoptosis analyses were carried out by flow cytometry. Protein expression was analyzed by immunoblotting. RESULTS: Nor-wogonin significantly inhibited the growth and decreased the viability of TNBC cells; however, it exhibited no or minimal effects in non-tumorigenic breast cells. Nor-wogonin (40 µM) was a more potent anti-proliferative and cytotoxic agent than wogonin (100 µM) and wogonoside (100 µM), which are structurally related to nor-wogonin. The antitumor effects of nor-wogonin can be attributed to cell cycle arrest via reduction of the expression of cyclin D1, cyclin B1, and CDK1. Furthermore, nor-wogonin induced mitochondrial apoptosis, (as evidenced by the increase in % of cells that are apoptotic), decreases in the mitochondrial membrane potential (ΔΨm), increases in Bax/Bcl-2 ratio, and caspase-3 cleavage. Moreover, nor-wogonin attenuated the expression of the nuclear factor kappa-B and activation of signal transducer and activator of transcription 3 pathways, which can be correlated with suppression of transforming growth factor-ß-activated kinase 1 in TNBC cells. CONCLUSION: These results showed that nor-wogonin might be a potential multi-target agent for TNBC treatment.

Self-encapsulation and controlled release of recombinant proteins using novel silica-forming peptides as fusion linkers.

Recently, the potential use of biomimetic silica as smart matrices for the auto-encapsulation and controlled release of functional proteins has gained increased interest because of the mild synthesis conditions. Inspired by biological silicification, in this study, we studied novel silica-forming peptides (SFPs), Volp1 and Salp1, to mediate the generation of silica hybrids in vitro. The fusion of SFPs to model fluorescent proteins directed their auto-encapsulation in wet sol-gel silica materials. Furthermore, the SFPs served as affinity linkers for the immobilization of recombinant proteins in silica. Interestingly, the SFP fusion proteins modulated silicic acid polycondensation and allowed for the self-immobilization of SFP fusion proteins in two distinct silica formulations depending on the ionic strength-precipitated silica particles or wet silica gel. The controlled release of Salp1/Volp1 fusion proteins from silica matrices was significantly greater than that of the silaffin R5 fusion proteins. Subsequently, we showed that multiple SFP-tagged proteins homogenously entrapped within a silica matrix could be separately released following pre-incubation with different concentrations of l-arginine solution. These new findings provide a simple and reproducible route for silica hybrid formation for in situ stable auto-encapsulation and the sustained release of recombinant proteins with potential applications in biotechnology.

Application of volcanic ash particles for protein affinity purification with a minimized silica-binding tag.

We recently reported that the spore coat protein, CotB1 (171 amino acids), from Bacillus cereus mediates silica biomineralization and that the polycationic C-terminal sequence of CotB1 (14 amino acids), designated CotB1p, serves as a silica-binding tag when fused to other proteins. Here, we reduced the length of this silica-binding tag to only seven amino acids (SB7 tag: RQSSRGR) while retaining its affinity for silica. Alanine scanning mutagenesis indicated that the three arginine residues in the SB7 tag play important roles in binding to a silica surface. Monomeric l-arginine, at concentrations of 0.3-0.5 M, was found to serve as a competitive eluent to release bound SB7-tagged proteins from silica surfaces. To develop a low-cost, silica-based affinity purification procedure, we used natural volcanic ash particles with a silica content of ∼70%, rather than pure synthetic silica particles, as an adsorbent for SB7-tagged proteins. Using green fluorescent protein, mCherry, and mKate2 as model proteins, our purification method achieved 75-90% recovery with ∼90% purity. These values are comparable to or even higher than that of the commonly used His-tag affinity purification. In addition to low cost, another advantage of our method is the use of l-arginine as the eluent because its protein-stabilizing effect would help minimize alteration of the intrinsic properties of the purified proteins. Our approach paves the way for the use of naturally occurring materials as adsorbents for simple, low-cost affinity purification.

The C-Terminal Zwitterionic Sequence of CotB1 Is Essential for Biosilicification of the Bacillus cereus Spore Coat.

UNLABELLED: Silica is deposited in and around the spore coat layer of Bacillus cereus, and enhances the spore's acid resistance. Several peptides and proteins, including diatom silaffin and silacidin peptides, are involved in eukaryotic silica biomineralization (biosilicification). Homologous sequence search revealed a silacidin-like sequence in the C-terminal region of CotB1, a spore coat protein of B. cereus. The negatively charged silacidin-like sequence is followed by a positively charged arginine-rich sequence of 14 amino acids, which is remarkably similar to the silaffins. These sequences impart a zwitterionic character to the C terminus of CotB1. Interestingly, the cotB1 gene appears to form a bicistronic operon with its paralog, cotB2, the product of which, however, lacks the C-terminal zwitterionic sequence. A ΔcotB1B2 mutant strain grew as fast and formed spores at the same rate as wild-type bacteria but did not show biosilicification. Complementation analysis showed that CotB1, but neither CotB2 nor C-terminally truncated mutants of CotB1, could restore the biosilicification activity in the ΔcotB1B2 mutant, suggesting that the C-terminal zwitterionic sequence of CotB1 is essential for the process. We found that the kinetics of CotB1 expression, as well as its localization, correlated well with the time course of biosilicification and the location of the deposited silica. To our knowledge, this is the first report of a protein directly involved in prokaryotic biosilicification. IMPORTANCE: Biosilicification is the process by which organisms incorporate soluble silicate in the form of insoluble silica. Although the mechanisms underlying eukaryotic biosilicification have been intensively investigated, prokaryotic biosilicification was not studied until recently. We previously demonstrated that biosilicification occurs in Bacillus cereus and its close relatives, and that silica is deposited in and around a spore coat layer as a protective coating against acid. The present study reveals that a B. cereus spore coat protein, CotB1, which carried a C-terminal zwitterionic sequence, is essential for biosilicification. Our results provide the first insight into mechanisms required for biosilicification in prokaryotes.

Affinity purification of recombinant proteins using a novel silica-binding peptide as a fusion tag.

We recently reported that silica is deposited on the coat of Bacillus cereus spores as a layer of nanometer-sized particles (Hirota et al. 2010 J Bacteriol 192: 111-116). Gene disruption analysis revealed that the spore coat protein CotB1 mediates the accumulation of silica (our unpublished results). Here, we report that B. cereus CotB1 (171 amino acids [aa]) and its C-terminal 14-aa region (corresponding to residues 158-171, designated CotB1p) show strong affinity for silica particles, with dissociation constants at pH 8.0 of 2.09 and 1.24 nM, respectively. Using CotB1 and CotB1p as silica-binding tags, we developed a silica-based affinity purification method in which silica particles are used as an adsorbent for CotB1/CotB1p fusion proteins. Small ubiquitin-like modifier (SUMO) technology was employed to release the target proteins from the adsorbed fusion proteins. SUMO-protease-mediated site-specific cleavage at the C-terminus of the fused SUMO sequence released the tagless target proteins into the liquid phase while leaving the tag region still bound to the solid phase. Using the fluorescent protein mCherry as a model, our purification method achieved 85 % recovery, with a purity of 95 % and yields of 0.60 ± 0.06 and 1.13 ± 0.13 mg per 10-mL bacterial culture for the CotB1-SUMO-mCherry and CotB1p-SUMO-mCherry fusions, respectively. CotB1p, a short 14-aa peptide, which demonstrates high affinity for silica, could be a promising fusion tag for both affinity purification and enzyme immobilization on silica supports.