Multifunctional Scaffolds Research Articles

Solvent and melting induced microspheres sintering techniques: a comparative study of morphology and mechanical properties

In this work we propose a bottom up approach founded on the assembly of building blocks by solvent induced microparticle sintering to realize multifunctional polymer scaffolds with predefined pore dimension and fully percolative pathway, able to include interspersing microdepot for the release of bioactive molecules. The aim of this study was to develop a versatile method of microspheres sintering based on the partial dissolution of the surface of adjacent microparticles and to compare it with melting induced microspheres sintering, just developed in a previous work. The two techniques were compared in terms of morphology, porosity and mechanical properties. The high potential of customizing the sintering process by the proper selection of the sintering techniques as well as microparticles with different features (e.g., material, size, shape, inner porosity) allows obtaining a wide pattern of micro/nanostructures with bio-inspired mechanical response so satisfying all basic requirements of a "smart" scaffold for bone tissue engineering.

Small Heat Shock Protein as a Multifunctional Scaffold: Integrated Tumor Targeting and Caspase Imaging within a Single Cage

Protein cages have the potential to serve as biomaterials for the targeted therapeutic and imaging systems. As an effort to exploit small heat shock protein (Hsp) cages as multifunctional biomaterials, we demonstrate that chemically and genetically modified Hsp cages permeate the cells via cancer cell binding and subsequent endocytic internalization and can image caspase activity in the live cells. Moreover, we report here that these functional Hsp cages can be specifically accumulated to tumor tissues of tumor-bearing mice when administered intravenously through the lateral tail vein. These tumor-targeting properties could be explained by the prolonged in vivo circulation and enhanced permeability and retention (EPR) effect as well as the ligand-mediated binding to cancer cells. Furthermore, when combined with the caspase sensing ability, our Hsp cage allows us to monitor the therapeutic evaluation after anticancer drug treatment by imaging the caspase activity within tumors. Therefore, we demonstrate that the Hsp cages have multifunctional scaffolds amenable to genetic and chemical modifications without loss of the cagelike architecture and can be exploited as biomedical materials including drug or imaging agent carriers.

Multifunctional magnetic mesoporous bioactive glass scaffolds with a hierarchical pore structure

Hyperthermia and local drug delivery have been proposed as potential therapeutic approaches for bone defects resulting from malignant bone tumors. The development of bioactive materials with magnetic and drug delivery properties may potentially meet this target. The aim of this study was to develop a multifunctional mesoporous bioactive glass (MBG) scaffold system for both hyperthermic and local drug delivery applications. To this end iron (Fe)-containing MBG (Fe-MBG) scaffolds with a hierarchical large pores structure (300–500 μm) and fingerprint-like mesopores (4.5 nm) have been prepared. The effects of Fe on the mesopore structure and physiochemical, magnetic, drug delivery and biological properties of MBG scaffolds have been systematically investigated. The results show that the morphology of the mesopores varied from straight channels to curved fingerprint-like channels after incorporation of Fe into MBG scaffolds. The magnetism of MBG scaffolds can be tailored by controlling the Fe content. Furthermore, the incorporation of Fe into mesoporous MBG glass scaffolds enhanced the mitochondrial activity and the expression of bone-related genes (ALP and OCN) in human bone marrow mesenchymal stem cells (BMSC) attached to the scaffolds. The Fe-MBG scaffolds obtained also possessed high specific surface areas and demonstrated sustained drug delivery. Thus Fe-MBG scaffolds are magnetic, degradable and bioactive. The multifunctionality of Fe-MBG scaffolds suggests that there is great potential for their use in the treatment and regeneration of large-bone defects caused by malignant bone tumors through a combination of hyperthermia, local drug delivery and osteoconductivity.

Targeting Oncogenic Protein-Protein Interactions by Diversity Oriented Synthesis and Combinatorial Chemistry Approaches

We are currently witnessing a decline in the development of efficient new anticancer drugs, despite the salient efforts made on all fronts of cancer drug discovery. This trend presumably relates to the substantial heterogeneity and the inherent biological complexity of cancer, which hinder drug development success. Protein-protein interactions (PPIs) are key players in numerous cellular processes and aberrant interruption of this complex network provides a basis for various disease states, including cancer. Thus, it is now believed that cancer drug discovery, in addition to the design of single-targeted bioactive compounds, should also incorporate diversity-oriented synthesis (DOS) and other combinatorial strategies in order to exploit the ability of multi-functional scaffolds to modulate multiple protein-protein interactions (biological hubs). Throughout the review, we highlight the chemistry driven approaches to access diversity space for the discovery of small molecules that disrupt oncogenic PPIs, namely the p53-Mdm2, Bcl-2/Bcl-xL-BH3, Myc-Max, and p53-Mdmx/Mdm2 interactions.

The pro-angiogenic properties of multi-functional bioactive glass composite scaffolds

The angiogenic properties of micron-sized (m-BG) and nano-sized (n-BG) bioactive glass (BG) filled poly(D,L lactide) (PDLLA) composites were investigated. On the basis of cell culture work investigating the secretion of vascular endothelial growth factor (VEGF) by human fibroblasts in contact with composite films (0, 5, 10, 20 wt %), porous 3D composite scaffolds, optimised with respect to the BG filler content capable of inducing angiogenic response, were produced. The in vivo vascularisation of the scaffolds was studied in a rat animal model and quantified using stereological analyses. The prepared scaffolds had high porosities (81–93%), permeability ( k = 5.4–8.6 × 10 −9 m 2) and compressive strength values (0.4–1.6 MPa) all in the range of trabecular bone. On composite films containing 20 wt % m-BG or n-BG, human fibroblasts produced 5 times higher VEGF than on pure PDLLA films. After 8 weeks of implantation, m-BG and n-BG containing scaffolds were well-infiltrated with newly formed tissue and demonstrated higher vascularisation and percentage blood vessel to tissue (11.6–15.1%) than PDLLA scaffolds (8.5%). This work thus shows potential for the regeneration of hard-soft tissue defects and increased bone formation arising from enhanced vascularisation of the construct.

Three-dimensional printing of hierarchical and tough mesoporous bioactive glass scaffolds with a controllable pore architecture, excellent mechanical strength and mineralization ability

New generation biomaterials for bone regeneration should be highly bioactive, resorbable and mechanically strong. Mesoporous bioactive glass (MBG), a novel bioactive material, has been used to study bone regeneration due to its excellent bioactivity, degradation and drug delivery ability, however, the construction of three-dimensional (3-D) MBG scaffolds (as for other bioactive inorganic scaffolds) for bone regeneration remains a significant challenge due to their inherent brittleness and low strength. In this brief communication we report a new facile method to prepare hierarchical and multifunctional MBG scaffolds with a controllable pore architecture, excellent mechanical strength and mineralization ability for application in bone regeneration by a modified 3-D printing technique using polyvinylalcohol (PVA) as a binder. The method provides a new way to solve commonly existing issues for inorganic scaffold materials, for example, uncontrollable pore architectures, low strength, high brittleness and the requirement for a second sintering at high temperature. The 3-D printed MBG scaffolds obtained possess a high mechanical strength about 200 times that of traditional polyurethane foam templated MBG scaffolds. They have a highly controllable pore architecture, excellent apatite mineralization ability and sustained drug delivery properties. Our study indicates that 3-D printed MBG scaffolds may be an excellent candidate for bone regeneration.

Multifunctional trackable dendritic scaffolds and delivery agents.

Dendrimers and other 3-D molecular assemblies are attractive scaffolds for biological delivery agents and diagnostic probes[1,2] due to their globular shape, modular structure, monodispersity and plurality of functional end groups.[3] To address this potential, a number of strategies and related dendritic architectures have been developed for delivery of bioactive molecules to desired cells or tissue,[4] with encapsulation[5] and covalent attachment to the dendritic chain ends being two major approaches.[6] While the encapsulation of drugs or dyes within the inner cavities of the dendrimer is promising,[5] in most cases only a limited number of guest molecules can be encapsulated even with dendrimers of high generations.[4d] Moreover, the non-covalent nature of the encapsulation makes it a challenge to control the stability of the loaded carrier and subsequent release of the payload.[4e] An alternative strategy exploits the large number of dendritic chain ends to carry the cargo molecules.[6] However, loading of large amounts of hydrophobic drugs or dyes can alter the dendrimer surface properties and decrease its solubility and bio-compatibility.[7] Partial functionalization[8] alleviates this issue but results in random chain end modification leading to a dispersity in loading, variable bio-performance and in many cases only low degrees of surface functionalization can be achieved without significantly changing the surface properties.[9]

Multifunctional Trackable Dendritic Scaffolds and Delivery Agents

Doppelfunktion: Multifunktionelle Dendrimere mit hoher innerer Beladungskapazität wurden an einem PEG-Kern aufgebaut. Die orthogonale Funktionalisierung der Kettenenden und inneren Gruppen ermöglicht sowohl die Markierung als auch die Beladung mit abspaltbaren Farbstoffen. Damit gelingt die simultane Verfolgung des Transports und der Freisetzung der Fracht in lebenden Zellen (siehe Bild). Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Shape-Memory Properties and Degradation Behavior of Multifunctional Electro-Spun Scaffolds

Multifunctional polymer-based biomaterials, which combine degradability and shape-memory capability, are promising candidate materials for the realization of active self-anchoring implants. In this work we explored the shape-memory capability as well as the hydrolytic and enzymatic in vitro degradation behavior of electro-spun scaffolds prepared from a multiblock copolymer, containing hydrolytically degradable poly(p-dioxanone) (PPDO) and poly(e-caprolactone) (PCL) segments, which we have named PDC. Electro-spun PDC scaffolds with an average deposit thickness of 80 ± 20 µm and a porosity in the range from 70% to 80% were prepared, where the single fiber diameter was around 3 µm. Excellent shape-memory properties were achieved with high recovery rate (Rr) values in the range of Rr = 92% to 98% and a recovery stress of smax = 4.6 MPa to 5.0 MPa. The switching temperature (Tsw) and the characteristic temperature obtained under constant strain recovery conditions (Ts,max) were found in the range from 32 °C to 35 °C, which was close to the melting temperature (Tm,PCL) associated to the poly(e-caprolactone) domains. A linear mass loss was observed in both hydrolytic and enzymatic degradation experiments. The mass loss was substantially accelerated, in enzymatic degradation when Pseudomonas cepacia lipase was added, which was reported to accelerate the degradation of PCL. During hydrolytic degradation a continuous decrease in elongation at break (eB) from eB = 800% to 15% was observed in a time period of 92 days, while in enzymatic degradation experiments a complete mechanical failure was obtained after 4 days.

Micropatterns of double-layered nanofiber scaffolds with dual functions of cell patterning and metabolite detection

This paper describes the development of multi-functional nanofiber scaffolds consisting of multiple layers of nanofiber scaffolds and nanofiber-incorporated poly(ethylene glycol) (PEG) hydrogels. As a proof-of-concept demonstration, we fabricated micropatterned polymeric nanofiber scaffolds that were capable of simultaneously generating cellular micropatterns within a biomimetic environment and detecting cellular metabolic products within well-defined microdomains. To achieve this goal, we designed nanofiber scaffolds with both vertical and lateral microdomains. Vertically heterogeneous structures that were responsible for multi-functionality were realized by preparing double-layered nanofiber scaffolds consisting of an antibody-immobilized bottom layer of nanofibers and an upper layer of bare polystyrene (PS) nanofibers by a two-step sequential electrospinning process. Photopatterning of poly(ethylene glycol) (PEG) hydrogel on the electrospun nanofibers produced laterally heterogeneous micropatterned nanofiber scaffolds made of hydrogel microwells filled with a nanofibrous region, which is capable of generating cell and protein micropatterns due to the different interactions that cells and proteins have with PEG hydrogels and nanofibers. When HepG2 cells were seeded into resultant nanofiber scaffolds, cells selectively adhered within the 200 μm × 200 μm PS fiber microdomain and formed 180.2 ± 6.7 μm spheroids after 5 days of culture in the upper layer. Furthermore, immobilized anti-albumin in the bottom layer detected albumin secreted by micropatterned HepG2 cells with higher sensitivity than flat PS substrates, demonstrating successful accomplishment of dual functions using micropatterned double-layered nanofiber scaffolds.

De novo designed positively charged tape-forming peptides: self-assembly and gelation in physiological solutions and their evaluation as 3D matrices for cell growth

Learning to control self-assembling nanostructures is currently one of the biggest challenges and promises in nanoscale science and nanotechnology. Nanostructured 3D matrices in particular are considered essential components in tissue engineering and regenerative medicine, e.g. as multifunctional scaffolds for cell encapsulation, growth and differentiation. Self-assembling peptide gels are a promising novel class of matrices for tissue engineering. Recently a new versatile family of negatively or positively charged tape-forming peptides have been de novo designed. These peptides were all found to self-assemble into nanostructured networks and gel cell transport medium in a simple, consistent and reproducible manner. Here we focus on the positively charged peptides of this family. We systematically changed the peptide to be amphiphilic or completely polar, or to be based on different polar uncharged amino acids (glutamine, serine, asparagine or threonine). The peptides were sterilised by γ-irradiation and were all found to be biocompatible using the contact cytotoxicity test. L929 murine fibroblast cells were encapsulated in 3D cell cultures inside 2% w/v gels and their proliferation was measured after 14 days using the ATP Lite assay. In this way a structure–function activity was established. Trifluoroacetic acid present in the peptide from the purification step was found to have a negative effect on cell proliferation. Peptide self-assembly in physiological conditions was studied extensively using spectroscopic and microscopic techniques, allowing rationalization of the observed biological structure–function activity. This detailed and systematic study enables us to develop refined criteria for the design of positively charged tape forming peptides and gels for biological and medical applications.

Multifunctional hybrid three-dimensionally woven scaffolds for cartilage tissue engineering.

The successful replacement of large-scale cartilage defects or osteoarthritic lesions using tissue-engineering approaches will likely require composite biomaterial scaffolds that have biomimetic mechanical properties and can provide cell-instructive cues to control the growth and differentiation of embedded stem or progenitor cells. This study describes a novel method of constructing multifunctional scaffolds for cartilage tissue engineering that can provide both mechanical support and biological stimulation to seeded progenitor cells. 3-D woven PCL scaffolds were infiltrated with a slurry of homogenized CDM of porcine origin, seeded with human ASCs, and cultured for up to 42 d under standard growth conditions. These constructs were compared to scaffolds derived solely from CDM as well as 3-D woven PCL fabric without CDM. While all scaffolds promoted a chondrogenic phenotype of the ASCs, CDM scaffolds showed low compressive and shear moduli and contracted significantly during culture. Fiber-reinforced CDM scaffolds and 3-D woven PCL scaffolds maintained their mechanical properties throughout the culture period, while supporting the accumulation of a cartilaginous extracellular matrix. These findings show that fiber-reinforced hybrid scaffolds can be produced with biomimetic mechanical properties as well as the ability to promote ASC differentiation and chondrogenesis in vitro.

Disposable amperometric biosensor based on nanostructured bacteriophages for glucose detection

The selection of electrode material profoundly influences biosensor science and engineering, as it heavily influences biosensor sensitivity. Here we propose a novel electrochemical detection method using a working electrode consisting of bio-nanowires from genetically modified filamentous phages and nanoparticles. fd-tet p8MMM filamentous phages displaying a three-methionine (MMM) peptide on the major coat protein pVIII (designated p8MMM phages) were immobilized on the active area of an electrochemical sensor through physical adsorption and chemical bonding. Bio-nanowires composed of p8MMM phages and silver nanoparticles facilitated sensitive, rapid and selective detection of particular molecules. We explored whether the composite electrode with bio-nanowires was an effective platform to detect the glucose oxidase. The current response of the bio-nanowire sensor was high at various glucose concentrations (0.1 µm–0.1 mM). This method provides a considerable advantage to demonstrate analyte detection over low concentration ranges. Especially, phage-enabled bio-nanowires can serve as receptors with high affinity and specificity for the detection of particular biomolecules and provide a convenient platform for designing site-directed multifunctional scaffolds based on bacteriophages and may serve as a simple method for label-free detection.

In vitro assembly of cubic RNA-based scaffolds designed in silico.

The organization of biological materials into versatile three-dimensional assemblies could be used to build multifunctional therapeutic scaffolds for use in nanomedicine. Here we report a strategy to design three-dimensional nanoscale scaffolds that can be self-assembled from RNA with precise control over their shape, size and composition. These cubic nanoscaffolds are only ~13 nm in diameter and are composed of short oligonucleotides making them amenable to chemical synthesis, point modifications and further functionalization. Nanocube assembly is verified by gel assays, dynamic light scattering and cryogenic electron microscopy. Formation of functional RNA nanocubes is also demonstrated by incorporation of a light-up fluorescent RNA aptamer that is optimally active only upon full RNA assembly. Moreover, we show the RNA nano-scaffolds can self-assemble in isothermal conditions (37°C) during in vitro transcription, which opens a route towards the construction of sensors, programmable packaging and cargo delivery systems for biomedical applications.

Effect of scaffold architecture and BMP-2/BMP-7 delivery on in vitro bone regeneration

The aim of this study was to develop 3-D tissue engineered constructs that mimic the in vivo conditions through a self-contained growth factor delivery system. A set of nanoparticles providing the release of BMP-2 initially followed by the release of BMP-7 were incorporated in poly(ε-caprolactone) scaffolds with different 3-D architectures produced by 3-D plotting and wet spinning. The release patterns were: each growth factor alone, simultaneous, and sequential. The orientation of the fibers did not have a significant effect on the kinetics of release of the model protein BSA; but affected proliferation of bone marrow mesenchymal stem cells. Cell proliferation on random scaffolds was significantly higher compared to the oriented ones. Delivery of BMP-2 alone suppressed MSC proliferation and increased the ALP activity to a higher level than that with BMP-7 delivery. Proliferation rate was suppressed the most by the sequential delivery of the two growth factors from the random scaffold on which the ALP activity was the highest. Results indicated the distinct effect of scaffold architecture and the mode of growth factor delivery on the proliferation and osteogenic differentiation of MSCs, enabling us to design multifunctional scaffolds capable of controlling bone healing.

Polymer-based composite scaffolds for tissue engineering.

Tissue engineering may be defined as the application of biological, chemical and engineering principles toward the repair, restoration or regeneration of living tissue using biomaterials, cells and biologically active molecules alone or in combinations. The rapid restoration of tissue biomechanical function represents a great challenge, highlighting the need to mimic tissue structure and mechanical behavior through scaffold designs. For this reason, several biodegradable and bioresorbable materials, as well as technologies and scaffold designs, have been widely investigated from an experimental and/or clinical point of view. Accordingly, this review aims at stressing the importance of polymer-based composite materials to make multifunctional scaffolds for tissue engineering, with a special focus on bone, ligaments, meniscus and cartilage. Moreover, polymer-based nanocomposites will also be briefly introduced as an interesting strategy to improve the biological and mechanical performances of polymer scaffolds, especially for bone tissue engineering.

Multifunctional Polymeric Scaffolds for Enhancement of PARACEST Contrast Sensitivity and Performance: Effects of Random Copolymer Variations

A DOTA (1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid) tetraamide ligand having a single acrylamide side-chain (M1) was copolymerized with either 2-methylacrylic acid (MAA), 2-(acryloylamino)-2-methyl-1-propanesulfonic acid (AMPS) or N-isopropylacrylamide (NIPAM) to create a series of linear random copolymers using classical free radical chain polymerization chemistry. The metal ion binding properties of hydrolyzed M1 were investigated by pH potentiometry and the europium (III) complexes of the resulting heteropolymers were evaluated as PARACEST imaging agents. All polymeric agents were found to possess similar intermediate-to-slow water exchange and CEST characteristics as the parent EuDOTA-tetraamide monomer. Consistent with basic multiplexing principles, the highest molecular weight polymer, Eu-DMAA 3.1, also showed the highest CEST sensitivity with a detection limit of 20 ± 2 μM. The second arylamide component gave polymers with widely different chemical characteristics and CEST properties. In particular, the Eu-DNIPAM 4.0 and Eu-DMAA 4.1 polymers displayed different solubility characteristics as a function of pH or temperature which, in turn, affected the water exchange and CEST properties of the corresponding agents. It was concluded that introduction of hydrophobic groups into the polymer backbone reduces solvent accessibility to the Eu(3+) component, effectively slowing water exchange between the inner-sphere water coordination position at each Eu(3+) center with bulk water. The CEST properties of the heteropolymers when dissolved in plasma suggest that the more hydrophobic characteristics of these polymers could be advantageous for in vivo applications.

Delivering Regenerative Cues to the Heart: Cardiac Drug Delivery by Microspheres and Peptide Nanofibers

Our understanding of signaling pathways and cues vital for cardiac regeneration is being refined by laboratories worldwide. As various mechanisms enabling cardiac regeneration are becoming elucidated, delivery vehicles suited for these potential therapeutics must also be developed. This review focuses on advances in two technologies, novel degradable microspheres for controlled release systems and self-assembling peptide nanofibers for cell and factor delivery. Polyketals, a new class of resorbable polymers, are well suited for treating inflammatory diseases due to biocompatible degradation products. Polyketals have been used to deliver small molecule inhibitors and antioxidant proteins to rat models of myocardial infarction with notable improvements in cardiac function. Self-assembling peptide nanofibers are a class of hydrogels that are well-defined scaffolds made up of 99% water and amenable to incorporation of a variety of bioactive cues. Work done by our laboratory and others have demonstrated functional improvements using these hydrogels as both a drug delivery vehicle for proteins as well as a defined microenvironment for transplanted cells. Combining non-inflammatory polymer microspheres for sustained release of drugs with self-assembling nanofibers yields multifunctional scaffolds that may soon drive the body's healing response following myocardial infarction towards cardiac regeneration.

Recombinant Gelatin Microspheres: Novel Formulations for Tissue Repair?

Microspheres (MSs) can function as multifunctional scaffolds in different approaches of tissue repair (TR), as a filler, a slow-release depot for growth factors, or a delivery vehicle for cells. Natural cell adhesion-supporting extracellular matrix components like gelatin are good materials for these purposes. Recombinant production of gelatin allows for on-demand design of gelatins, which is why we aim at developing recombinant gelatin (RG) MSs for TR. Two types of MSs (50 < Ø < 100 microm) were prepared by crosslinking two RGs, Syn-RG, and the arginine-glycine-aspartate-containing Hu-RG. The MSs were characterized, and their tissue reaction and degradation in rats was examined. Histological analysis of the explants after 14 and 28 days in vivo also showed that Syn-RG was degraded slower than Hu-RG, which correlated with the in vitro degradation assay. Hu-RG explants displayed more cellular ingrowth (60% vs. 15% for Syn-RG at day 14), which was associated with extracellular matrix deposition and vascularization. The infiltrating cells consisted of mainly macrophages, part of which fused to giant cells locally, and fibroblasts. No differences were found in matrix metalloproteinase mRNA levels, whereas gelatinase activity was clearly higher in Hu-RG explants. In conclusion, the in vitro and in vivo results of these novel formulations pave the way for cell- and/or factor-driven TR by these RG MSs.

Hybrid 3D structure of poly( d, l-lactic acid) loaded with chitosan/chondroitin sulfate nanoparticles to be used as carriers for biomacromolecules in tissue engineering

In the tissue engineering (TE) field, the concept of producing multifunctional scaffolds, capable not only of acting as templates for cell transplantation but also of delivering bioactive agents in a controlled manner, is an emerging strategy aimed to enhance tissue regeneration. In this work, a complex hybrid release system consisting in a three-dimensional (3D) structure based on poly( d, l-lactic acid) (PDLLA) impregnated with chitosan/chondroitin sulfate nanoparticles (NPs) was developed. The scaffolds were prepared by supercritical fluid foaming at 200 bar and 35 °C, and were then characterized by scanning electron microscopy (SEM) and micro-CT. SEM also allowed to assess the distribution of the NPs within the structure, showing that the particles could be found in different areas of the scaffold, indicating a homogeneous distribution within the 3D structure. Water uptake and weight loss measurements were also carried out and the results obtained demonstrated that weight loss was not significantly enhanced although the entrapment of the NPs in the 3D structure clearly enhances the swelling of the structure. Moreover, the hybrid porous biomaterial displayed adequate mechanical properties for cell adhesion and support. The possibility of using this scaffold as a multifunctional material was further evaluated by the incorporation of a model protein, bovine serum albumin (BSA), either directly into the PDLLA foam or in the NPs that were eventually included in the scaffold. The obtained results show that it is possible to achieve different release kinetics, suggesting that this system is a promising candidate for dual protein delivery system for TE applications.