Exploring Protein-Based Carriers in Drug Delivery: A Review.

Drug delivery systems (DDSs) represent an emerging focus for many researchers and they are becoming progressively crucial in the development of new treatments. Great attention is given to all the challenges that a drug has to overcome during its journey across barriers and tissues and all the pharmacokinetics modulations that are needed in order to reach the targeting sites. The goal of these pathways is the delivery of drugs in a controlled way, optimizing their bioavailability and minimizing side effects. Recent innovations in DDSs include various nanotechnology-based approaches, such as nanoparticles, nanofibers and micelles, which provide effective targeted delivery and sustained release of therapeutics. In this context, protein-based drug delivery systems are gaining significant attention in the pharmaceutical field due to their potential to revolutionize targeted and efficient drug delivery. As natural biomolecules, proteins offer distinct advantages, including safety, biocompatibility and biodegradability, making them a fascinating alternative to synthetic polymers. Moreover, protein-based carriers, including those derived from gelatin, albumin, collagen, gliadin and silk proteins, demonstrate exceptional stability under physiological conditions, and they allow for controlled and sustained drug release, enhancing therapeutic efficacy. This review provides a comprehensive overview of the current trends, challenges, and future perspectives in protein-based drug delivery, focusing on the types of proteins adopted and the techniques that are being developed to enhance their functionality in terms of drug affinity and targeting capabilities, underscoring their potential to significantly impact modern therapeutics.

Mutation in the Bombyx mori BmGMC2 gene impacts silk production and silk protein synthesis.

Silk is a natural protein fiber that is predominantly comprised of fibroin and sericin. In addition, it contains seroins, protease inhibitors, enzymes, and other proteins. We found an ecdysone oxidase BmGMC2, notably, which is specifically and highly expressed only in the silk glands of silkworms (Bombyx mori L.). It is also one of the main components of non-cocoon silk, however, its precise function remains unclear. In this study, we examined the spatiotemporal expression pattern of this protein and obtained a homozygous mutant strain (K-GMC2) using the CRISPR-Cas9 system. Compared to the wild-type strain (WT), the silk production and main silk proteins significantly decreased in the larval stage, and the adhesive strength of native silk proteins decreased in the final instar. Proteomic data indicated the abundance of ribosomal proteins decreased significantly in K-GMC2, differentially expressed proteins (DEPs) were enriched in pathways related to neurodegenerative diseases and genetic information processing, indicating that knockout may lead to a certain degree of cell stress, affecting the synthesis of silk proteins. This study investigated the expression pattern and gene function of ecdysone oxidase BmGMC2 in silk and silk glands, laying the groundwork for understanding the role of enzymes in the production of silk fibers.

Membrane-based inverse-transition purification facilitates a rapid isolation of various spider-silk elastin-like polypeptide fusion proteins from extracts of transgenic tobacco.

Plants can produce complex pharmaceutical and technical proteins. Spider silk proteins are one example of the latter and can be used, for example, as compounds for high-performance textiles or wound dressings. If genetically fused to elastin-like polypeptides (ELPs), the silk proteins can be reversibly precipitated from clarified plant extracts at moderate temperatures of ~ 30 °C together with salt concentrations > 1.5 M, which simplifies purification and thus reduces costs. However, the technologies developed around this mechanism rely on a repeated cycling between soluble and aggregated state to remove plant host cell impurities, which increase process time and buffer consumption. Additionally, ELPs are difficult to detect using conventional staining methods, which hinders the analysis of unit operation performance and process development. Here, we have first developed a surface plasmon resonance (SPR) spectroscopy-based assay to quantity ELP fusion proteins. Then we tested different filters to prepare clarified plant extract with > 50% recovery of spider silk ELP fusion proteins. Finally, we established a membrane-based purification method that does not require cycling between soluble and aggregated ELP state but operates similar to an ultrafiltration/diafiltration device. Using a data-driven design of experiments (DoE) approach to characterize the system of reversible ELP precipitation we found that membranes with pore sizes up to 1.2 µm and concentrations of 2-3 M sodium chloride facilitate step a recovery close to 100% and purities of > 90%. The system can thus be useful for the purification of ELP-tagged proteins produced in plants and other hosts.

Sustainable Silk-Based Particulate Systems for the Controlled Release of Pharmaceuticals and Bioactive Agents in Wound Healing and Skin Regeneration.

The increasing demand for innovative approaches in wound healing and skin regeneration has prompted extensive research into advanced biomaterials. This review focuses on showcasing the unique properties of sustainable silk-based particulate systems in promoting the controlled release of pharmaceuticals and bioactive agents in the context of wound healing and skin regeneration. Silk fibroin and sericin are derived from well-established silkworm production and constitute a unique biocompatible and biodegradable protein platform for the development of drug delivery systems. The controlled release of therapeutic compounds from silk-based particulate systems not only ensures optimal bioavailability but also addresses the challenges associated with conventional delivery methods. The multifaceted benefits of silk proteins, including their inherent biocompatibility, versatility, and sustainability, are explored in this review. Furthermore, the intricate mechanisms by which controlled drug release takes place from silk-based carriers are discussed.

Review of Spider Silk Applications in Biomedical and Tissue Engineering.

This review will present the latest research related to the production and application of spider silk and silk-based materials in reconstructive and regenerative medicine and tissue engineering, with a focus on musculoskeletal tissues, and including skin regeneration and tissue repair of bone and cartilage, ligaments, muscle tissue, peripheral nerves, and artificial blood vessels. Natural spider silk synthesis is reviewed, and the further recombinant production of spider silk proteins. Research insights into possible spider silk structures, like fibers (1D), coatings (2D), and 3D constructs, including porous structures, hydrogels, and organ-on-chip designs, have been reviewed considering a design of bioactive materials for smart medical implants and drug delivery systems. Silk is one of the toughest natural materials, with high strain at failure and mechanical strength. Novel biomaterials with silk fibroin can mimic the tissue structure and promote regeneration and new tissue growth. Silk proteins are important in designing tissue-on-chip or organ-on-chip technologies and micro devices for the precise engineering of artificial tissues and organs, disease modeling, and the further selection of adequate medical treatments. Recent research indicates that silk (films, hydrogels, capsules, or liposomes coated with silk proteins) has the potential to provide controlled drug release at the target destination. However, even with clear advantages, there are still challenges that need further research, including clinical trials.

Superfast Gelation of Spider Silk-Based Artificial Silk Protein.

Spider silk proteins (spidroins) have garnered attention in biomaterials research due to their ability to self-assemble into hydrogels. However, reported spidroin hydrogels require high protein concentration and prolonged gelation time. Our study engineered an artificial spidroin that exhibits unprecedented rapid self-assembly into hydrogels at physiologically relevant conditions, achieving gelation at a low concentration of 6 mg/mL at 37 °C without external additives. Remarkably, at a 30 mg/mL concentration, our engineered protein forms hydrogels within 30 s, a feature we termed "superfast gelation". This rapid formation is modulated by ions, pH, and temperature, offering versatility in biomedical applications. The hydrogel's capacity to encapsulate proteins and support E. coli growth while inducing RFP expression provides a novel platform for drug delivery and bioengineering applications. Our findings introduce a superfast, highly adaptable, and cytocompatible hydrogel that self-assembles under mild conditions, underscoring the practical implication of rapid gelation in biomedical research and clinical applications.

Factors Influencing Properties of Spider Silk Coatings and Their Interactions within a Biological Environment.

Biomaterials are an indispensable part of biomedical research. However, although many materials display suitable application-specific properties, they provide only poor biocompatibility when implanted into a human/animal body leading to inflammation and rejection reactions. Coatings made of spider silk proteins are promising alternatives for various applications since they are biocompatible, non-toxic and anti-inflammatory. Nevertheless, the biological response toward a spider silk coating cannot be generalized. The properties of spider silk coatings are influenced by many factors, including silk source, solvent, the substrate to be coated, pre- and post-treatments and the processing technique. All these factors consequently affect the biological response of the environment and the putative application of the appropriate silk coating. Here, we summarize recently identified factors to be considered before spider silk processing as well as physicochemical characterization methods. Furthermore, we highlight important results of biological evaluations to emphasize the importance of adjustability and adaption to a specific application. Finally, we provide an experimental matrix of parameters to be considered for a specific application and a guided biological response as exemplarily tested with two different fibroblast cell lines.

A comprehensive review of recent advances in silk sericin: Extraction approaches, structure, biochemical characterization, and biomedical applications.

Silks are natural polymers that have been widely used for centuries. Silk consists of a filament core protein, termed fibroin, and a glue-like coating substance formed of sericin (SER) proteins. This protein is extracted from the silkworm cocoons (particularly Bombyx mori) and is mainly composed of amino acids like glycine, serine, aspartic acid, and threonine. Silk SER can be obtained using numerous methods, including enzymatic extraction, high-temperature, autoclaving, ethanol precipitation, cross-linking, and utilizing acidic, alkali, or neutral aqueous solutions. Given the versatility and outstanding properties of SER, it is widely fabricated to produce sponges, films, and hydrogels for further use in diverse biomedical applications. Hence, many authors reported that SER benefits cell proliferation, tissue engineering, and skin tissue restoration thanks to its moisturizing features, antioxidant and anti-inflammatory properties, and mitogenic effect on mammalian cells. Remarkably, SER is used in drug delivery depending on its chemical reactivity and pH-responsiveness. These unique features of SER enhance the bioactivity of drugs, facilitating the fabrication of biomedical materials at nano- and microscales, hydrogels, and conjugated molecules. This review thoroughly outlines the extraction techniques, biological properties, and respective biomedical applications of SER.

Analysis of histomorphometric and proteome dynamics inside the silk gland lumen of Bombyx mori revealed the dynamic change of silk protein during the molt stage.

Silkworms spin different silks at different growth stages for specific purposes. The silk spun before the end of each instar is stronger than that at the beginning of each instar and cocoon silk. However, the compositional changes in silk proteins during this process are unknown. Consequently, we performed histomorphological and proteomic analyses of the silk gland to characterize changes from the instar end to the next instar beginning. The silk glands were collected on day 3 of third- and fourth-instar larvae (III-3 and IV-3) and the beginning of fourth-instar larvae (IV-0). Proteomic analysis identified 2961 proteins from all silk glands. Silk proteins P25 and Ser5 were significantly more abundant in III-3 and IV-3 than in IV-0, and many cuticular proteins and protease inhibitors increased significantly in IV-0 compared with III-3 and IV-3. This shift may cause mechanical property differences between the instar end and beginning silk. Using section staining, qPCR, and western blotting, we found for the first time that silk proteins were degraded first and then resynthesized during the molting stage. Furthermore, we revealed that fibroinase mediated the changes of silk proteins during molting. Our results provide insights into the molecular mechanisms of silk proteins dynamic regulation during molting.

Characteristic Evaluation of Recombinant MiSp/Poly(lactic-co-glycolic) Acid (PLGA) Nanofiber Scaffolds as Potential Scaffolds for Bone Tissue Engineering.

Biomaterial-based nanofibrous scaffolds are the most effective alternative to bone transplantation therapy. Here, two recombinant minor ampullate spidroins (spider silk proteins), R1SR2 and NR1SR2C, were blended with Poly(lactic-co-glycolic) Acid (PLGA), respectively, to generate nanofiber scaffolds by electrospinning. The N-terminal (N), C-terminal (C), repeating (R1 and R2) and spacer (S) modules were all derived from the minor ampullate spidroins (MiSp). The physical properties and structures of the blended scaffolds were measured by scanning electron microscopy (SEM), water contact angle measurement, Fourier transform infrared spectroscopy (FTIR), Differential scanning calorimetry (DSC), and Tensile mechanical testing. The results showed that blending of MiSp (R1SR2 and NR1SR2C) reduced the diameter of nanofibers, increased the porosity and glass transition temperatures of nanofibrous scaffolds, and effectively improved the hydrophilicity and ultimate strain of scaffolds. It is worth noting that the above changes were more significant in the presence of the N- and C-termini of MiSp. In cell culture assays, human bone mesenchymal stem cells (HBMSCs) grown on NR1SR2C/PLGA (20/80) scaffolds displayed markedly enhanced proliferative and adhesive abilities compared with counterparts grown on pure PLGA scaffolds. Jointly, these findings indicated recombinant MiSp/PLGA, particularly NR1SR2C/PLGA (20/80) blend nanofibrous scaffolds, is promising for bone tissue engineering.

Structure-Property Relationship Based on the Amino Acid Composition of Recombinant Spider Silk Proteins for Potential Biomedical Applications.

Improving biomaterials by engineering application-specific and adjustable properties is of increasing interest. Most of the commonly available materials fulfill the mechanical and physical requirements of relevant biomedical applications, but they lack biological functionality, including biocompatibility and prevention of microbial infestation. Thus, research has focused on customizable, application-specific, and modifiable surface coatings to cope with the limitations of existing biomaterials. In the case of adjustable degradation and configurable interaction with body fluids and cells, these coatings enlarge the applicability of the underlying biomaterials. Silks are interesting coating materials, e.g., for implants, since they exhibit excellent biocompatibility and mechanical properties. Herein, we present putative implant coatings made of five engineered recombinant spider silk proteins derived from the European garden spider Araneus diadematus fibroins (ADF), differing in amino acid sequence and charge. We analyzed the influence of the underlying amino acid composition on wetting behavior, blood compatibility, biodegradability, serum protein adsorption, and cell adhesion. The outcome of the comparison indicates that spider silk coatings can be engineered for explicit biomedical applications.

Design and Fabrication of a Dual Protein-Based Trilayered Nanofibrous Scaffold for Efficient Wound Healing.

Chronic wound healing is a major threat all over the world. There are currently a plethora of biomaterials-based wound dressings available for wound healing applications. In this study, a dual protein-based (silk fibroin and sericin) nanofibrous scaffold from a natural source (B.mori silkworm cocoons) with antibacterial and antioxidative properties for wound healing was investigated. An electrospun layer-by-layer silk protein-based nanofibrous scaffold was fabricated with a top layer of hydrophobic silk fibroin protein blended with polyvinyl alcohol (PVA), a middle layer of waste protein silk sericin loaded with silver(I) sulfadiazine as an antibacterial agent, and a bottom layer using silk fibroin blended with polycaprolactone (PCL). The trilayered nanofibrous scaffold with a smooth and bead-free morphology demonstrated excellent wettability, slow in vitro degradation, controlled drug release, and potent antibacterial and antioxidant properties. In vitro, the scaffold also demonstrated excellent hemocompatibility and biocompatibility. Furthermore, in vivo wound contraction, histological, and micro-CT investigations show complete wound healing and the formation of new skin tissue in a male Balb/c mouse model treated with the scaffold. The antioxidant properties of the sericin protein and SSD-based triple-layered nanofibrous scaffold protect the wound from bacterial infection and improve wound healing in a mouse model. The current study develops a dual protein-based nanofibrous scaffold with antibacterial and antioxidant properties as a promising wound dressing material.

Proteomic characterization of the fibroin-based silk fibers produced by weaver ant Camponotus textor.

The fibroin-based silk fibers of weaver ants are an alternative biomaterial to be investigated and explored for potential biomedical applications. In this context, the silk fibers from the nest of the weaver ant Camponotus textor was solubilized and fractionated by gel permeation. The different fractions were collected, pooled and submitted to analysis with a series of biochemical methods, nuclear magnetic resonance (NMR) spectroscopy, analytical proteomic strategies, and data treatment with bioinformatic tools to perform the structural characterization of the fibroin-based silk fibers produced by the ant. Our data demonstrated the identification of one fibroin proteoform in the ant silk fibers. The protein chracterized as a glycoprotein with MW around 40 kDa and presenting 66% (w/w) of total sugars attached to it through O-linked carbohydrates. The 3D of protein was modeled, revealing a structure predominantly constituted of coiled-coil secondary units in the whole model, featuring at least four superhelices (arrangement with multiple α-helices). The scientific outcomes reported herein may be relevant for the development of novel approaches for the synthetic or recombinant production of novel silk-based polymers for biomedical applications. BIOLOGICAL SIGNIFICANCE: The present investigation significantly expanded knowledge regarding to the fibroin-based silk fibers from weaver ants, contributing to improvements in our understanding of the properties and characteristics of these silk fibers. For example, as reported here, carbohydrates were detected in the ants' silk for the first time presenting the fibroin as a glycoprotein. Moreover, the 3D structure provided new insights into the secondary structures considering the whole model of the protein.

Cloning, expression, and characterization of a novel sericin-like protein.

Silk consists of two proteins called fibroin and sericin. While fibroin is used in the textile industry and has various biomaterial applications, sericin has been considered as waste material until recently. Sericin is a multicomponent protein and it has important properties such as biocompatibility, biodegradability, cryoprotectivity, and antioxidant. Sericin from silkworm cocoons can be obtained by chemical, enzymatic, and heat treatment methods. However, sericin obtained with these treatment methods is not of consistent and high quality. Moreover, the exposure of sericin to harsh conditions during extraction leads to inconsistencies in the composition and structure of the sericin obtained. The inconsistencies in sericin structure and composition decrease application of sericin as a biomaterial. Here, we produce a sericin-like protein (Ser4mer) with native sequence of sericin encoding four repeats of the conserved 38 amino acid motif recombinantly in Escherichia coli and characterize its structural properties. Ser4mer protein shows similar structure to native sericin and higher solubility than previously obtained recombinant sericin-like proteins. Recombinant production of a soluble sericin-like protein will significantly expand its applications as a biomaterial. In addition, recombinant production of silk proteins will allow us to understand sequence-structure relationships in these proteins.

Prevention of chemically induced hair damage by means of treatment based on proteins and polysaccharides.

BACKGROUND: There is currently a great interest not only in developing products for the protection and recovery of chemically damaged hair, but also in developing effective protocols to investigate the impact of chemical treatments and attest the efficacy of innovative hair care products. Among the most relevant cosmetic treatments for hair are bleaching and coloring, which have been shown to significantly impair mechanical and structural properties. OBJECTIVES: This study aimed to characterize the damage induced by hair bleaching and coloring and to evaluate the protective effects of a hair care treatment based on integral silk proteins (fibroin and sericin) and vegetable-derived polysaccharides from linseed (Linum usitatissimum L.). METHODS: Hair swatches were subjected to different treatment protocols in order to evaluate the protective effect of proposed and benchmark products during bleaching and coloring processes. Tensile tests were performed to assess mechanical properties and improvement in resistance to breakage. Goniophotometric measurements were applied to determine improvement in luster. Hair fiber surface and relief were evaluated by SEM image analysis. RESULTS: Swatches bleached and treated with both evaluated products had a significant increase in resistance and reduced structural damage. Swatches colored and treated with both evaluated products showed reduced structural damage, and a significant increase in resistance and luster after the 1st and 5th washes. CONCLUSIONS: The proposed product was effective in protecting and repairing bleached and colored swatches, improving resistance and luster and reducing structural damage. By applying complementary techniques within a reliable evaluation protocol, it was possible to attest the protective properties of the product under study.

Silk Microneedle Patch Capable of On-Demand Multidrug Delivery to the Brain for Glioblastoma Treatment.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Surgery followed by chemotherapy and radiotherapy remains the standard treatment strategy for GBM patients. However, challenges still exist when surgery is difficult or impossible to remove the tumor completely. Herein, the design, fabrication and application of a heterogenous silk fibroin microneedle (SMN) patch is reported for circumventing the blood-brain barrier and releasing multiple drugs directly to the tumor site for drug combination treatment. The biocompatible and biodegradable SMN patch can dissolve slowly over time, allowing the sustained release of multiple drugs at different doses. Furthermore, it can be triggered remotely to induce rapid drug delivery at a designated stage after implantation. In the GBM mouse models, two clinically relevant chemotherapeutic agents (thrombin and temozolomide) and targeted drug (bevacizumab) are loaded into the SMN patch with individually controlled release profiles. The drugs are spatiotemporally and sequentially delivered for hemostasis, anti-angiogenesis, and apoptosis of tumor cells. Device application is non-toxic and results in decreased tumor volume and increased survival rate in mice. The SMN patch with on-demand multidrug delivery has potential applications for the combined administration of therapeutic drugs for the clinical treatment of brain tumors when other methods are insufficient.

Immune Response to Silk Sericin-Fibroin Composites: Potential Immunogenic Elements and Alternatives for Immunomodulation.

The unique properties of silk proteins (SPs), particularly silk sericin (SS) and silk fibroin (SF), have attracted attention in the design of scaffolds for tissue engineering over the past decades. Since SF has good mechanical properties, while SS displays bioactivity, scaffolds combining both proteins should exhibit complementary properties enhancing the potential of these materials. Unfortunately, SS-SF composites can generate chronic immune responses and their immunogenic element is not completely clear. The potential of SS-SF composites in tissue engineering, elements which may contribute to their immunogenicity, and alternatives for their preparation and design, to modulate the immune response and take advantage of their useful properties, are discussed in this review. It is known that SS can enhance ß-sheet formation in SF, which may act as hydrophobic regions with a strong affinity for adsorption proteins inducing the chronic recruitment of inflammatory cells. Therefore, tailoring the exposure of hydrophobic regions at the scaffold surface should represent a viable strategy to modulate the immune response. This can be achieved by coating SS-SF composites with SS or other hydrophilic polymers, to take advantage of their antibiofouling properties. Research is still needed to realize the full potential of these composites for tissue engineering.

Biomaterials for Soft Tissue Repair and Regeneration: A Focus on Italian Research in the Field.

Tissue repair and regeneration is an interdisciplinary field focusing on developing bioactive substitutes aimed at restoring pristine functions of damaged, diseased tissues. Biomaterials, intended as those materials compatible with living tissues after in vivo administration, play a pivotal role in this area and they have been successfully studied and developed for several years. Namely, the researches focus on improving bio-inert biomaterials that well integrate in living tissues with no or minimal tissue response, or bioactive materials that influence biological response, stimulating new tissue re-growth. This review aims to gather and introduce, in the context of Italian scientific community, cutting-edge advancements in biomaterial science applied to tissue repair and regeneration. After introducing tissue repair and regeneration, the review focuses on biodegradable and biocompatible biomaterials such as collagen, polysaccharides, silk proteins, polyesters and their derivatives, characterized by the most promising outputs in biomedical science. Attention is pointed out also to those biomaterials exerting peculiar activities, e.g., antibacterial. The regulatory frame applied to pre-clinical and early clinical studies is also outlined by distinguishing between Advanced Therapy Medicinal Products and Medical Devices.

Multifunctional and Ultrathin Electronic Tattoo for On-Skin Diagnostic and Therapeutic Applications.

Epidermal electronic systems for detecting electrophysiological signals, sensing, therapy, and drug delivery are at the frontier in man-machine interfacing for healthcare. However, it is still a challenge to develop multifunctional bioapplications with minimal invasiveness, biocompatibility, and stable electrical performance under various mechanical deformations of biological tissues. In this study, a natural silk protein with carbon nanotubes (CNTs) is utilized to realize an epidermal electronic tattoo (E-tattoo) system for multifunctional applications that address these challenging issues through dispersing highly conductive CNTs onto the biocompatible silk nanofibrous networks with porous nature to construct skin-adhesive ultrathin electronic patches. Individual components that incorporate electrically and optically active heaters, a temperature sensor (temperature coefficient of resistance of 5.2 × 10-3  °C-1 ), a stimulator for drug delivery (>500 µm penetration depth in skin), and real-time electrophysiological signal detectors are described. This strategy of E-tattoos integrated onto human skin can open a new route to a next-generation electronic platform for wearable and epidermal bioapplications.

Native-like Flow Properties of an Artificial Spider Silk Dope.

Recombinant spider silk has emerged as a biomaterial that can circumvent problems associated with synthetic and naturally derived polymers, while still fulfilling the potential of the native material. The artificial spider silk protein NT2RepCT can be produced and spun into fibers without the use of harsh chemicals and here we evaluate key properties of NT2RepCT dope at native-like concentrations. We show that NT2RepCT recapitulates not only the overall secondary structure content of a native silk dope but also emulates its viscoelastic rheological properties. We propose that these properties are key to biomimetic spinning and that optimization of rheological properties could facilitate successful spinning of artificial dopes into fibers.