Recent Progress in Protein-Polyphenol Assemblies for Biomedical Applications.

Nowadays, natural materials as smart building blocks for assembling functional materials have aroused extensive interest in the scientific community. Proteins and polyphenols are typical natural building blocks that are widely used. On the one hand, proteins are one of the most versatile classes of biomolecules, serving as catalysts, signaling molecules, transporters, receptors, scaffolds that maintain the integrity of cell and tissue, and more. On the other hand, the facile adhesion of naturally abundant polyphenols with other substances and their potential biomedical applications have been highly attractive for functional biomaterials fabrication. Additionally, there are a variety of interactions between the proteins and polyphenols, mainly hydrogen bonding, hydrophobic, and ionic interactions. These reversible dynamic interactions enable proteins and polyphenols to form stable protein-polyphenol assemblies and maintain their inherent structures and biological activities in the assemblies. Therefore, protein-polyphenol assemblies can be applied to design a variety of advanced functional materials for biomedical applications. Herein, recent progress in protein-polyphenol particles, capsules, coatings, and hydrogels is summarized, the preparation and application of these assemblies are introduced in detail, and the future of the field is prospected.

Hydroxyapatite Film with Distinctive Roughness for Simulating the Bone Microenvironment and Revealing the Behavior of Metastatic Mammary Cancer.

Breast cancer is a common malignant tumor arising in normal mammary epithelial tissues. Nearly 75% of the patients with advanced mammary cancer develop bone metastases, resulting in secondary tumor growth, osteolytic bone degradation, and poor prognosis. The bone matrix comprises a highly hierarchical architecture and is composed of a nonmineral organic part, a predominantly type-I collagen, and a mineral inorganic part composed of hydroxyapatite (HA) nanocrystals (Ca10(PO4)6(OH)2). Although there has been extensive research indicating that the material properties of bone minerals affect metastatic breast cancer, it remains unclear how the microenvironment of the bone matrix, such as the roughness, which changes as a result of osteolytic bone remodeling, affects this disease. In this study, we created HA coatings in situ on polyelectrolyte multilayers (PEMs) by incubating PEMs in a mixture of phosphate and calcium ions. The HA films with distinctive roughness were successfully collected by controlling the incubation time, which served as the simulated microenvironment of the bone matrix. MDA-MB231 breast cancer cells were cultured on HA films, and an optimal roughness was observed in the adhesion, proliferation, and expression of two cytokines closely related to bone metastasis. This study contributed to the understanding of the effect of the microenvironment of the bone matrix, such as the roughness, on the metastasis behavior of breast cancer.

Injectable, Self-Healing and Multiple Responsive Histamine Modified Hyaluronic Acid Hydrogels with Potentialities in Drug Delivery, Antibacterial and Tissue Engineering.

Hydrogels are 3D network structures composed of physically or chemically crosslinked, hydrophilic molecules. Compared with conventional hydrogels with static and permanent network structures, injectable and responsive hydrogels generated from dynamic networks, have attracted increasing attention from various disciplines due to their wide-ranging applications in tissue engineering, drug delivery, soft robotics, etc. Herein, an injectable self-healing and multiple-responsive hyaluronic acid (HA)- histamine (His)/metal hydrogel is developed by modifying His onto HA and the subsequent, dynamic coordination between imidazole and metal ions. The pH-responsive and mechanical behaviors exhibited by the HA-His/metal hydrogels are tunable with the kinds and the concentrations of metal ions. The HA-His/Zr4+ hydrogels demonstrate a moldable capability at a neutral pH and a multi-stimulus-responsive capability when exposed to a weak alkaline environment and hyaluronidase, which inhibits bacterial growth and biofilm formation. Biocompatibilities and accelerated wound healing are demonstrated in vitro and in vivo and are thoroughly investigated and well characterized. The HA-His/Zr4+ hydrogel has great potential in various biomedical applications, such as pH- and hyaluronidase-responsive sustained release, antibacterial, and implantable materials for tissue engineering.

Material priority engineered metal-polyphenol networks: mechanism and platform for multifunctionalities.

Engineering the surface of materials with desired multifunctionalities is an effective way to fight against multiple adverse factors during tissue repair process. Recently, metal-polyphenol networks (MPNs) have gained increasing attention because of their rapid and simple deposition process onto various substrates (silicon, quartz, gold and polypropylene sheets, etc.). However, the coating mechanism has not been clarified, and multifunctionalized MPNs remain unexplored. Herein, the flavonoid polyphenol procyanidin (PC) was selected to form PC-MPN coatings with Fe3+, and the effects of different assembly parameters, including pH, molar ratio between PC and Fe3+, and material priority during coating formation, were thoroughly evaluated. We found that the material priority (addition sequence of PC and Fe3+) had a great influence on the thickness of the formed PC-MPNs. Various surface techniques (e.g., ultraviolet-visible spectrophotometry, quartz crystal microbalance, X-ray photoelectron spectroscopy, atomic force microscopy, and scanning electron microscopy) were used to investigate the formation mechanism of PC-MPNs. Then PC-MPNs were further engineered with multifunctionalities (fastening cellular attachment in the early stage, promoting long-term cellular proliferation, antioxidation and antibacterial activity). We believe that these findings could further reveal the coating formation mechanism of MPNs and guide the future design of MPN coatings with multifunctionalities, thereby greatly broadening their application prospects, such as in sensors, environments, drug delivery, and tissue engineering.

Secondary Structure-Dominated Layer-by-Layer Growth Mode of Protein Coatings.

Benefiting from the luxury functions of proteins, protein coatings have been extended to various applications, including tissue engineering scaffolds, drug delivery, antimicrobials, sensing and diagnostic equipment, food packaging, etc. Fast construction of protein coatings is always interesting to materials science and significant to industrialization. Here, we report a layer-by-layer (LbL) multilayer-constructed coating of tannic acid (TA) and lysozyme (Lyz), in which the secondary conformations of Lyz dominate the growth rate of the TA/Lyz coating. As well characterized by various techniques (quartz crystal microbalance with dissipation (QCM-D), circular dichroism (CD) spectra, Fourier transform infrared (FTIR) spectroscopy, atomic force microscopy (AFM), contact angle, etc.), TA-induced conformational transition of Lyz to α-helices occurs at pH 8 from other secondary structures (ß-sheets, ß-turns, and random coils), which leads to the very fast growth of TA/Lyz with a number of deposited bilayers, with thicknesses of more than 90 nm for six bilayers. In contrast to the leading conformation of α-helices at pH 8, Lyz displayed multiple conformations (α-helices, ß-sheets, ß-turns, and random coils) at pH 6, which resulted in coating thicknesses of less than 30 nm for six bilayers. By the addition of NaCl, Tween 20, and urea, we further confirmed that the secondary conformations of Lyz relied greatly on the interactions between TA and Lyz and dominated the growth rate of the multilayers. We believe that these findings will help to understand the transformation of secondary conformations by TA or other polyphenols and inspire a new route to quickly build protein coatings.

Silanization of a Metal-Polyphenol Coating onto Diverse Substrates as a Strategy for Controllable Wettability with Enhanced Performance to Resist Acid Corrosion.

Wettability is a crucial characteristic of materials that plays a vital role in surface engineering. Surface modification is the key to changing the wettability of materials, and a simple and universal modification approach is being extensively pursued by researchers. Recently, metal-phenolic networks (MPNs) have been widely studied because they impart versatility and functionality in surface modification. However, an MPN is not stable for long periods, especially under acidic conditions, and is susceptible to pollution by invasive species. Spurred by the versatility of MPNs and various functionalities achieved by silanization, we introduce a general strategy to fabricate functionally stable coatings with controllable surface wettability by combining the two methods. The formation process of MPN and silane-MPN coatings was characterized by spectroscopic ellipsometry (SE), UV-visible-near-infrared (UV-vis-NIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), atomic force microscopy (AFM), water contact angle (WCA), etc. We found that the stability of the MPN was greatly enhanced after silanization, which is attributed to the cross-linking effect that occurs between silane and the MPN, namely, the cross-linking protection produced in this case. Additionally, the wettability of an MPN can be easily changed through our strategy. We trust that our strategy can further extend the applications of MPNs and points toward potential prospects in surface modification.

One-Pot Generating Subunit Vaccine with High Encapsulating Efficiency and Fast Lysosome Escape for Potent Cellular Immune Response.

Utilizing nanoparticles to deliver subunit vaccine is considered to be a promising strategy to improve immune response. However, currently reported systems suffered from one or more points, for example, delicate design on molecular structures and elaborate synthesis process, low antigen and/or adjuvant encapsulation efficiency, involvement of toxic materials, and denaturing of bioactivity of antigen and/or adjuvant. To address these issues, here, for the first time, we developed a one-pot method to produce a subunit vaccine by using hexa-histidine metal assembly (HmA) to codeliver tumor-associated antigens (GP100, a peptide KTWGQYWQV) and adjuvant (CpG). The generation of subunit vaccines was detailedly characterized by various techniques, including dynamic scatter, scanning electron microscopy, transmission electron microscopy, UV-visible spectroscopy, agarose gel electrophoresis, etc. HmA displayed high efficiency on encapsulating both subunits (GP100 and CpG) under mild conditions, and the generated subunit vaccine showed a pH-dependent release profile of loaded subunits. In the cellular tests, these subunit vaccines behaved with a quick endocytosis into immune cells and a fast endo/lysosomes escape, inducing maturation of antigen presentative cells and stimulating a potent cellular immune response. These results suggested that HmA is a robust platform for fabricating subunit vaccine, with immense potential for the immunotherapy of various diseases.

Hexahistidine-Metal Assemblies: A Facile and Effective Codelivery System of Subunit Vaccines for Potent Humoral and Cellular Immune Responses.

Fully effective vaccines must induce both potent humoral and cellular immunities. Nanoparticles coencapsulating antigens and adjuvants have shown promising advantages as subunit vaccines in many aspects. However, the low loading efficiency and complicated synthesis process of these nanomaterials need to be improved. Here, we utilized hexahistidine (His6)-metal assembly (HmA) particles as carriers to codeliver ovalbumin peptides and cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs). We found that antigen/adjuvant-carrying HmA can efficiently enter into antigen-presenting cells and help the antigens escape from lysosomes to induce the maturation of these cells in vitro, characterized by increasing expression levels of costimulatory molecules and cytokines. More importantly, the vaccines with high biocompatibility can elicit strong humoral and cellular immunities by improving secretion of specific antibodies and cytokines, enhancing activation of DCs and T cells in vivo. Our results suggest that HmA provides a new approach for subunit vaccines by codelivery of antigens and adjuvants.

Shape Effect of Nanoparticles on Tumor Penetration in Monolayers Versus Spheroids.

The physical properties of nanoparticles (NPs), such as size, surface chemistry, elasticity, and shape, have exerted a profound influence on tumor penetration. However, the effect of shape on cellular uptake and tumor penetration is still unclear because of the different chemical compositions and shapes of tested particles and the use of inapposite cellular models. To discover the effect of NP shapes on cellular uptake and tumor penetration and bridge the gap between models in vivo and in vitro, elongated polystyrene (PS) NPs with a fixed volume, an identical chemical composition, and the same zeta potential, but with different aspect ratios (ARs), were generated. The physical properties, cellular uptake, tumor penetration, and corresponding mechanisms of these NPs were thoroughly investigated. We discovered that the elongated PS particles with higher ARs had lower uptake rates in the 2-dimensional cell monolayer culture model in vitro, but they showed optimal ARs in the evaluated three-dimensional spheroid model. Although the elongated PS particles had a similar tumor penetration mechanism (mainly through extracellular pathways), the percentage of penetration using these mechanisms was strongly dependent on the ARs. As an alternative model for studies in vivo, spheroids were used instead of the cell monolayer for the development of drug delivery systems. In addition, the physicochemical properties of NPs must be delicately balanced and adjusted to achieve the best therapeutic outcomes.

Graphene Oxide and Lysozyme Ultrathin Films with Strong Antibacterial and Enhanced Osteogenesis.

There is a great demand worldwide for bone-related implant materials. The drawbacks of chronic infections and poor bone healing of current implant materials have limited their clinical applications. Functionalizing the implant surfaces with antibacterial and osteogenic films on implant materials provides new opportunities for fabricating novel implant materials. In the present study, an ultrathin (GO/Lys)8 film of several tens of nanometers was fabricated using a layer-by-layer (LBL) technique with alternative deposition of graphene oxide (GO) and lysozyme (Lys). The deposition of the (GO/Lys) n film exhibited a successive growth as supported by ellipsometry, UV-vis, and Fourier transform infrared data, and the physical properties (morphology, roughness, and stiffness) of this film were characterized with an atomic force microscope. The ultrathin films exhibited a great effect on bacterium sterilization of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli and enhanced osteogenic differentiation efficiency, showing the potential application in bone implant coatings. We believe that this LBL assembling strategy will pave the way for fabricating dual-functional surfaces and guide the design of the implanted surfaces in the future.

Improved Ozonation Efficiency for Polymerization Mother Liquid from Polyvinyl Chloride Production Using Tandem Reactors.

Polymerization mother liquid (PML) is one of the main sources of wastewater in the chlor-alkali industry. The effective degradation of the PML produced in PVC polymerization using three or five ozone reactors in tandem was designed with a focus on improving the ozonation efficiency. The ozonation efficiency of the tandem reactors for the degradation of PML, along with the effect of ozone concentration, the number of reactors utilized in series, and the reaction time on the chemical oxygen demand (COD) removal were investigated in detail. The results showed that the COD removal increased as the ozone concentration was increased from 10.6 to 60 mg·L-1, achieving 66.4% COD removal at ozone concentration of 80.6 mg·L-1. However, when the ozone concentration was increased from 60 mg·L-1 to 80 mg·L-1, the COD removal only increased very little. The COD decreased with increasing ozone concentration. During the initial degradation period, the degradation rate was the highest at both low and high ozone concentrations. The degradation rate decreased with reaction time. The rate at a low ozone concentration decreased more significantly than at high ozone concentration. Although high ozone concentration is desirable for COD removal and degradation rate, the utilization efficiency of ozone decreased with increasing ozone concentration. The ozone utilization efficiency of the five-reactor device was three times higher than that of three tandem reactors, demonstrating that ozonation utilization efficiency can be improved by increasing the number of tandem reactors. Ozonation in tandem reactors is a promising approach for PML treatment.

Effect of Roughness on in Situ Biomineralized CaP-Collagen Coating on the Osteogenesis of Mesenchymal Stem Cells.

Because of its outstanding osteo-conductive property, a calcium phosphate (CaP) coating has been used as an implant coating for bone tissue engineering. Nevertheless, the issues, such as harsh fabrication conditions, long-term stability and biocompatibility, and the requirement for expensive instruments, still exist in current coating techniques. To address these issues, the CaP coatings doped with collagen (CaP-Col) were in situ generated on polyelectrolyte multilayers (PEMs) by incubating PEMs in a mixture of the collagen, phosphate, and calcium ions. The resulting coatings have controllable physical properties (chemical composition, crystallinity, and roughness) and good stability before and after incubation with cell culture medium. We also found that both the cellular viability and osteogenesis of mesenchymal stem cells (MSCs) were closely related to the roughness of PEMs/CaP-Col, one of the easily ignored physical factors in current coating designs but very critical. The existed roughness window (between 18 ± 1.2 and 187 ± 7.3 nm) suitable for MSC proliferation on PEMs/CaP-Col coating and the optimal roughness (∼98 ± 3.5 nm) for MSC osteogenesis further demonstrated that the roughness was a critical factor for bone formation. Therefore, we envision that our exploration of the effects of surface roughness on MSC behaviors would provide better guidance for the future design of material coating and eventual medical success.

Engineering Polyelectrolyte Capsules with Independently Controlled Size and Shape.

Polyelectrolyte capsules (PECs) are a promising delivery system that has the ability to carry a large payload of a variety of cargoes. Controlling PEC properties is critical to understanding and tuning their cellular uptake efficiency, kinetics, and mechanism as well as their biodistribution in the body. The lack of a method to independently engineer PEC size, shape, and chemistry impedes both basic understanding of how physicochemical parameters affect PEC behavior in drug delivery and other applications, and the ability to optimize parameters for best function. Here, we report the successful fabrication of PECs having constant surface chemistry with independently controlled size and shape by combining soft organic templates created by the particle stretching method and a modified layer-by-layer (LBL) deposition process. Changing the template dispersion solution during LBL deposition from water to ethanol allowed us to overcome previous issues with organic templates, such as aggregation and template removal. These results will contribute not only to the basic study of the role of capsule shape and size on its function but also to the optimization of capsule properties for drug or imaging carriers, sensors, reactors, and other applications.

Genetically Engineered Plant Viral Nanoparticles Direct Neural Cells Differentiation and Orientation.

An important aim of tissue engineering is to design biomimetic materials with specific cell binding motifs and precisely controlled structural organization, thereby providing biochemical and physical cues for desired cellular behaviors. Previously, our group generated genetically modified tobacco mosaic virus (TMV) displaying integrin binding motifs, RGD1, RGD7, PSHRN3, P15, and DGEA. The resulting rod-like virus particles displaying integrin binding motifs were biocompatible with Neuro 2A (N2a), a mouse neural crest-derived cell line, and could promote the neurite outgrowth of N2a. The genetically modified viruses could be assembled with aligned orientation in the capillary by applying a shear force. The resulting aligned substrates were able to dictate directional neurite outgrowth of N2a cells. Therefore, this method could be potentially applied for neural tissue engineering, as a neural conduit for repairing peripheral nerve injuries.

Aligned Electroactive TMV Nanofibers as Enabling Scaffold for Neural Tissue Engineering.

Electroactive nanofibers were fabricated by in situ polymerization of aniline on the surface of tobacco mosaic virus (TMV) using sodium poly(styrenesulfonate) (PSS) as dopant. These electroactive TMV/PANi/PSS nanofibers were employed to support growth of neuronal cells, resulting in augmentation of the length of neurites. In addition, the percentage of cells with neurites was increased in comparison to cells cultured on TMV-derived nonconductive nanofibers. The TMV-based electroactive nanofibers could be aligned in capillaries that could guide the outgrowth direction of neurites, increase the percentage of cells with neurites, and lead to a bipolar cellular morphology. Our results demonstrate that the electroactivity and topographical cues provided by TMV/PANi/PSS nanofibers can synergistically stimulate neural cells differentiation and neurites outgrowth, which make it a promising scaffolding material for neural tissue engineering.

Optimization of boronic ester-based amphiphilic copolymers for ROS-responsive drug delivery.

This study introduces boronic ester-based ROS-responsive amphiphilic copolymers for antioxidant drug delivery. Tuning the hydrophobic/hydrophilic balance optimized the size, curcumin encapsulation, ROS-triggered release, cellular uptake, and intracellular ROS scavenging. The lead P1b formulation self-assembled into stable 10 nm micelles enabling rapid ROS-triggered curcumin release and preferential cellular internalization. P1b eliminated over 90% of pathogenic intracellular ROS within 10 minutes, demonstrating a rapid antioxidant therapy.

Gelatin-based dynamic response antioxidant, anti-inflammatory multifunctional hydrogel for enhanced diabetic wound repair.

Diabetic wound therapy presents significant challenges in the clinical environment, where persistent bleeding, disturbed inflammatory regulation, impaired cellular proliferation, and impaired tissue remodeling are major features of diabetic wound healing. However, current treatment strategies need to be considered in the context of the dynamic and complex needs of chronic wound healing. Here, multifunctional dynamic boronic acid cross-linked hydrogels were prepared by the reaction of gelatin (Gel) inoculated with 5-carboxy 3-nitrophenylboronic acid (NPBA) and Epigallocatechin gallate (EGCG) to achieve rapid gelation at pH = 7.4, EGCG could interact electrostatically with cationic antimicrobial peptides (AMP) to achieve the effective loading of AMP in the hydrogels. This hydrogel can be injected and adhered to skin defects in diabetic patients to provide a barrier and rapid hemostasis. In a high glucose microenvironment, the rapid release of AMP effectively kills bacteria, while the responsive release of EGCG eliminates reactive oxygen species (ROS) and promotes macrophage M2 polarization. In addition, the hydrogel had excellent biocompatibility and degradability properties, degraded completely after 3 days of subcutaneous injection, and was non-toxic in H&E staining of major organs and serum liver function indices in mice. This multifunctional injectable hydrogel accelerates diabetic skin wound repair and is a promising dressing for the precise treatment of diabetic wounds.

Presenting dual-functional peptides on implant surface to direct in vitro osteogenesis and in vivo osteointegration.

The complex biological process of osseointegration and the bio-inertness of bone implants are the major reasons for the high failure rate of long-term implants, and have also promoted the rapid development of multifunctional implant coatings in recent years. Herein, through the special design of peptides, we use layer-by-layer assembly technology to simultaneously display two peptides with different biological functions on the implant surface to address this issue. A variety of surface characterization techniques (ellipsometry, atomic force microscopy, photoelectron spectroscopy, dissipation-quartz crystal microbalance) were used to study in detail the preparation process of the dual peptide functional coating and the physical and chemical properties, such as the composition, mechanical modulus, stability, and roughness of the coating. Compared with single peptide functional coatings, dual-peptide functionalized coatings had much better performances on antioxidant, cellular adhesion in early stage, proliferation and osteogenic differentiation in long term, as well as in vivo osteogenesis and osseointegration capabilities. These findings will promote the development of multifunctional designs in bone implant coatings, as a coping strategy for the complexity of biological process during osteointegration.

Enhanced Ocular Delivery of Beva via Ultra-Small Polymeric Micelles for Noninvasive Anti-VEGF Therapy.

Pathological ocular neovascularization resulting from retinal ischemia constitutes a major cause of vision loss. Current anti-VEGF therapies rely on burdensome intravitreal injections of Bevacizumab (Beva). Herein ultrasmall polymeric micelles encapsulating Beva (P@Beva) are developed for noninvasive topical delivery to posterior eye tissues. Beva is efficiently loaded into 11 nm micelles fabricated via self-assembly of hyperbranched amphiphilic copolymers. The neutral, brush-like micelles demonstrate excellent drug encapsulation and colloidal stability. In vitro, P@Beva enhances intracellular delivery of Beva in ocular cells versus free drug. Ex vivo corneal and conjunctival-sclera-choroidal tissues transport after eye drops are improved 23-fold and 7.9-fold, respectively. Anti-angiogenic bioactivity is retained with P@Beva eliciting greater inhibition of endothelial tube formation and choroid sprouting over Beva alone. Remarkably, in an oxygen-induced retinopathy (OIR) model, topical P@Beva matching efficacy of intravitreal Beva injection, is the clinical standard. Comprehensive biocompatibility verifies safety. Overall, this pioneering protein delivery platform holds promise to shift paradigms from invasive intravitreal injections toward simplified, noninvasive administration of biotherapeutics targeting posterior eye diseases.

Responsively Degradable Nanoarmor-Assisted Super Resistance and Stable Colonization of Probiotics for Enhanced Inflammation-Targeted Delivery.

Manipulation of the gut microbiota using oral microecological preparations has shown great promise in treating various inflammatory disorders. However, delivering these preparations while maintaining their disease-site specificity, stability, and therapeutic efficacy is highly challenging due to the dynamic changes associated with pathological microenvironments in the gastrointestinal tract. Herein, a superior armored probiotic with an inflammation-targeting capacity is developed to enhance the efficacy and timely action of bacterial therapy against inflammatory bowel disease (IBD). The coating strategy exhibits suitability for diverse probiotic strains and has negligible influence on bacterial viability. This study demonstrates that these armored probiotics have ultraresistance to extreme intraluminal conditions and stable mucoadhesive capacity. Notably, the HA-functionalized nanoarmor equips the probiotics with inflamed-site targetability through multiple interactions, thus enhancing their efficacy in IBD therapy. Moreover, timely "awakening" of ingested probiotics through the responsive transferrin-directed degradation of the nanoarmor at the site of inflammation is highly beneficial for bacterial therapy, which requires the bacterial cells to be fully functional. Given its easy preparation and favorable biocompatibility, the developed single-cell coating approach provides an effective strategy for the advanced delivery of probiotics for biomedical applications at the cellular level.