One of the most effective methods to treat cancer is the specific delivery of anticancer drugs to the target site. To achieve this goal, we designed an anticancer drug with mild hyperthermia-mediated triggering and tumor-specific delivery. To enhance the thermosensitive drug release, we incorporated elastin-like polypeptide (ELP), which is known to be a thermally responsive phase transition peptide into the dipalmitoylphosphatidylcholine (DPPC)-based liposome surface. Additionally, cyclic arginine-glycine-aspartic acid (cRGD) binds to alphavbeta3 integrin, which is overexpressed in angiogenic vasculature and tumor cells, was introduced on the liposome. ELP-modified liposomes with the cRGD targeting moiety were prepared using a lipid film hydration method, and doxorubicin (DOX) was loaded into the liposome by the ammonium sulfate-gradient method. The cRGD-targeted and ELP-modified DOX-encapsulated liposomes (RELs) formed spherical vesicles with a mean diameter of 181 nm. The RELs showed 75% and 83% DOX release at 42 degrees C and 45 degrees C, respectively. The stability of RELs was maintained up to 12h without the loss of their thermosensitive function for drug release. Flow cytometry results showed that the cellular uptake of DOX in RELs into alphavbeta3 integrin-overexpressing U87MG and HUVEC cells was 8-fold and 10-fold higher, respectively, than that of non-targeting liposomes. Confocal microscopy revealed that REL released DOX only under the mild hyperthermia condition at 42 degrees C by showing the localization of DOX in nuclei and the liposomes in the cytosol. The cell cytotoxicity results demonstrated that REL can efficiently kill U87MG cells through cRGD targeting and thermal triggering. The in vivo tumoral accumulation measurement showed that the tumor-targeting effect of RELs was 5-fold higher than that of non-targeting liposomes. This stable, target-specific, and thermosensitive liposome shows promise to enhance therapeutic efficacy if it is applied along with a relevant external heat-generating medical system.

C-X-C chemokine receptor type 4 (CXCR4) is an alpha-chemokine receptor specific for stromal cell-derived factor 1 (SDF-1 also called CXCL12). The antagonist of CXCR4 can mobilize CD34+ cells and hematopoietic stem cells from bone marrow within several hours, and it has an efficacy on diabetes ulcer through acting on the SDF-1/CXCR4 axis. In this study, we investigated for the first time whether the antagonist of CXCR4 (Plerixafor/AMD3100) delivered on acellular dermal matrix (ADM) may accelerate diabetes-impaired wound healing. ADM scaffolds were fabricated from nondiabetic mouse skin through decellularization processing and incorporated with AMD3100 to construct ADM-AMD3100 scaffold. Full-thickness cutaneous wound in streptozotocin (STZ)-induced diabetic mice were treated with ADM, AMD3100, or ADM-AMD3100. 21 days after treatment, wound closure in ADM-AMD3100-treated mice was more complete than ADM group and AMD3100 group, and it was accompanied by thicker collagen formation. Correspondingly, diabetic mice treated with ADM-AMD3100 demonstrated prominent neovascularization (higher capillary density and vascular smooth muscle actin), which were accompanied by up-regulated mRNA levels of SDF-1 and enhanced migration of CXCR4 in the granulation tissue. Our results demonstrate that ADM scaffold provide perfect niche for loading AMD3100 and ADM-AMD3100 is a promising method for diabetic wound healing mainly by increasing expression of SDF-1 and enhancing migration of CXCR4-positive cells.

Nanofiber wound dressings have great potential for both acute and chronic wound healing. The aim of this study is to develop a wound dressing by the electrospinning method and to determine its in vitro characteristics. The viscosity and the surface tension values of the polymer solutions used in the electrospinning were measured and their suitability for electrospinning was evaluated. Nanofiber wound dressing consists of three layers. The first and the second layers are sodium alginate and chitosan nanofibers, respectively. The core of the coaxial nanofibers that comprises the third layer of the wound dressing contains 1% polycaprolactone and 4.5% collagen, the shell comprises 2.5% doxycycline and 2.5% polyethylene oxide. The developed wound dressing comprises aligned nanofibers, with a contact angle of 38 degrees , a work of bioadhesion value of 0.485mJ/cm(2) on rat skin, a tensile strength of 2.76MPa, an elongation at break value of 7.65%, a specific surface area of 9.65m(2)/g and a porosity of 52.3%. The amount of doxycycline content was found to be 260mug/cm(2) and the complete drug release was achieved in 15min. No cytotoxic effect was shown in cell culture studies with keratinocyte cell lines. As a result of the stability studies, it was found that the morphological, mechanical, bioadhesion and wettability properties and the amount of doxycycline remained stable for a period of 12 months at 4 degrees C/ambient humidity condition. The developed three-layered wound dressing could be an alternative for wound healing applications.

One of the major applications of tissue-engineered skin substitutes for wound healing is to promote the healing of cutaneous wounds. In this respect, many important clinical milestones have been reached in the past decades. However, currently available skin substitutes for wound healing often suffer from a range of problems including wound contraction, scar formation, and poor integration with host tissue. Engineering skin substitutes by tissue engineering approach has relied upon the creation of three-dimensional scaffolds as extracellular matrix (ECM) analog to guide cell adhesion, growth, and differentiation to form skin-functional and structural tissue. The three-dimensional scaffolds can not only cover wound and give a physical barrier against external infection as wound dressing, but also can provide support both for dermal fibroblasts and the overlying keratinocytes for skin tissue engineering. A successful tissue scaffold should exhibit appropriate physical and mechanical characteristics and provide an appropriate surface chemistry and nano and microstructures to facilitate cellular attachment, proliferation, and differentiation. A variety of scaffolds have been fabricated based on materials ranging from naturally occurring ones to those manufactured synthetically. This review discusses a variety of commercial or laboratory-engineered skin substitutes for wound healing. Central to the discussion are the scaffolds/materials, fabrication techniques, and their characteristics associated with wound healing. One specifically highlighted emerging fabrication technique is electrospinning that allows the design and fabrication of biomimetic scaffolds that offer tremendous potential applications in wound healing of skin.

Collagen is the primary component of the extracellular matrix in the human body. It has proved challenging to fabricate collagen scaffolds capable of replicating the structure and function of tissues and organs. We present a method to 3D-bioprint collagen using freeform reversible embedding of suspended hydrogels (FRESH) to engineer components of the human heart at various scales, from capillaries to the full organ. Control of pH-driven gelation provides 20-micrometer filament resolution, a porous microstructure that enables rapid cellular infiltration and microvascularization, and mechanical strength for fabrication and perfusion of multiscale vasculature and tri-leaflet valves. We found that FRESH 3D-bioprinted hearts accurately reproduce patient-specific anatomical structure as determined by micro-computed tomography. Cardiac ventricles printed with human cardiomyocytes showed synchronized contractions, directional action potential propagation, and wall thickening up to 14% during peak systole.

Human civilization has witnessed the use of materials-derived from biomolecules of plants and animal origin for biomedical applications since ancient era. In recent years, precision design principles have been adopted to develop novel biomaterials derived from biomolecules. The biomolecules-derived biomaterials fabrication is dependent on chemical, biochemical and mechanical parameters of biomolecules and their bulk materials. Thus, structural variations and weak noncovalent interactions present within the basic building blocks greatly influence the functional features and applications. This comprehensive review provides one-stop information on recent innovations of various biomaterial-types derived from a diverse class of biomolecules through selected and representative examples with potential biomedical applications ranging from diagnosis, biosensing, antimicrobial efficacy, anticancer therapeutics, drug delivery, bioprinting, bioimaging, tissue engineering and regenerative medicine. The discussion systematically follows the top-down approach in the order of molecular complexity viz., biomacromolecules, oligomers and monomers of all classes of biomolecules (proteins, nucleic acids, carbohydrates and lipids) including a special section on biohybrid materials derived from molecular systems integrated with more than one class of biomolecules. In addition to providing overview of impressive advancements in the area, synergistic integration of biomolecules with synthetic materials to develop smart biomaterials is emphasized to improve the chemical, mechanical, stimuli-responsiveness, immunogenicity and biocompatibility features.

Multiphoton microscopy of collagen hydrogels produces second harmonic generation (SHG) and two-photon fluorescence (TPF) images, which can be used to noninvasively study gel microstructure at depth ( approximately 1 mm). The microstructure is also a primary determinate of the mechanical properties of the gel; thus, we hypothesized that bulk optical properties (i.e., SHG and TPF) could be used to predict bulk mechanical properties of collagen hydrogels. We utilized polymerization temperature (4-37 degrees C) and glutaraldehyde to manipulate collagen hydrogel fiber diameter, space-filling properties, and cross-link density. Multiphoton microscopy and scanning electron microscopy reveal that as polymerization temperature decreases (37-4 degrees C) fiber diameter and pore size increase, whereas hydrogel storage modulus (G', from 23 +/- 3 Pa to 0.28 +/- 0.16 Pa, respectively, mean +/- SE) and mean SHG decrease (minimal change in TPF). In contrast, glutaraldehyde significantly increases the mean TPF signal (without impacting the SHG signal) and the storage modulus (16 +/- 3.5 Pa before to 138 +/- 40 Pa after cross-linking, mean +/- SD). We conclude that SHG and TPF can characterize differential microscopic features of the collagen hydrogel that are strongly correlated with bulk mechanical properties. Thus, optical imaging may be a useful noninvasive tool to assess tissue mechanics.

Synthetic VGGVG, a

Elastin-like polypeptides (ELPs) undergo a reversible, inverse phase transition. Below their transition temperature (Tt), ELPs are soluble in water, but when the temperature is raised above Tt, phase transition occurs, leading to aggregation of the polypeptide. We demonstrate a method for purification of soluble fusion proteins incorporating an ELP tag. Advantages of this method, termed

Elastin-like polypeptides (ELPs) are recombinant protein domains exhibiting lower critical solution temperature (LCST) behavior. This LCST behavior is controlled not only by intrinsic factors including amino acid composition and polypeptide chain length but also by non-ELP fusion domains. Here, we report that the presence of a composite non-ELP sequence that includes both His and T7 tags or a short Ser-Lys-Gly-Pro-Gly (SKGPG) sequence can dramatically change the LCST behavior of a positively-charged ELP domain. Both the His and T7 tags have been widely used in recombinant protein design to enable affinity chromatography and serve as epitopes for protein detection. The SKGPG sequence has been used to improve the expression of ELPs. Both the composite tag and the SKGPG sequence are <15% of the total length of the ELP fusion proteins. Despite the small size of the composite tag, its incorporation imparted pH-sensitive LCST behavior to the positively-charged ELP fusion protein. This pH sensitivity was not observed with the incorporation of the SKGPG sequence. The pH sensitivity results from both electrostatic and hydrophobic interactions between the composite tag and the positively-charged ELP domain. The hydrophobicity of the composite tag also alters the ELP interaction with Hofmeister salts by changing the overall hydrophobicity of the fusion protein. Our results suggest that incorporation of short tag sequences should be considered when designing temperature-responsive ELPs and provide insights into utilizing both electrostatic and hydrophobic interactions to design temperature-responsive recombinant proteins as well as synthetic polymers.

In nature, organic matrix macromolecules play a critical role in enhancing the mechanical properties of biomineralized composites such as bone and teeth. Designing artificial matrix analogues is promising but challenging because relatively little is known about how natural matrix components function. Therefore, in lieu of using natural components, we created biomimetic matrices using genetically engineered elastin-like polypeptides (ELPs) and then used them to construct mechanically robust ELP-hydroxyapatite (HAP) composites. ELPs were engineered with well-defined backbone charge distributions by periodic incorporation of negative, positive, or neutral side chains or with HAP-binding octaglutamic acid motifs at one or both protein termini. ELPs exhibited sequence-specific capacities to interact with ions, bind HAP, and disperse HAP nanoparticles. HAP-binding ELPs were incorporated into calcium phosphate cements, resulting in materials with improved mechanical strength, injectability, and antiwashout properties. The results demonstrate that rational design of genetically engineered polymers is a powerful system for determining sequence-property relationships and for improving the properties of organic-inorganic composites. Our approach may be used to further develop novel, multifunctional bone cements and expanded to the design of other advanced composites.

Here we present the design of an engineered, elastin-like protein (ELP) that is chemically modified to enable stable coatings on the surfaces of titanium-based dental and orthopaedic implants by novel photocrosslinking and solution processing steps. The ELP includes an extended RGD sequence to confer bio-signaling and an elastin-like sequence for mechanical stability. ELP thin films were fabricated on cp-Ti and Ti6Al4V surfaces using scalable spin and dip coating processes with photoactive covalent crosslinking through a carbene insertion mechanism. The coatings withstood procedures mimicking dental screw and hip replacement stem implantations, a key metric for clinical translation. They promoted rapid adhesion of MG63 osteoblast-like cells, with over 80% adhesion after 24 h, compared to 38% adhesion on uncoated Ti6Al4V. MG63 cells produced significantly more mineralization on ELP coatings compared to uncoated Ti6Al4V. Human bone marrow mesenchymal stem cells (hMSCs) had an earlier increase in alkaline phosphatase activity, indicating more rapid osteogenic differentiation and mineral deposition on adhesive ELP coatings. Rat tibia and femur in vivo studies demonstrated that cell-adhesive ELP-coated implants increased bone-implant contact area and interfacial strength after one week. These results suggest that ELP coatings withstand surgical implantation and promote rapid osseointegration, enabling earlier implant loading and potentially preventing micromotion that leads to aseptic loosening and premature implant failure.

We developed rubber-like elastomeric materials using a natural elastin derived sequence and genetic engineering to create precisely defined elastin-like polypeptides. The coiled elastin-like polypeptide chains, which behave like entropic springs, were cross-linked using an end-to-end tethering scheme to synthesize simple hydrogels with excellent extensibility and reversibility. Our hydrogels extend to strains as high as 1500% and remain highly resilient with elastic recovery as high as 94% even at 600% strain, significantly exceeding any other protein-based hydrogel. These materials are valuable as elastomeric hydrogels for designing extremely robust scaffolds useful for tissue engineering.

As an alternative to natural extracellular matrix (ECM) macromolecules, cell-adhesion peptides (CAPs) have had tremendous impact on the design of cell culture platforms, implants, and wound dressings. However, only a handful of CAPs have been utilized. The discrepancy in ECM composition strongly affects cell behavior, so it is paramount to reproduce such differences in synthetic systems. This Opinion article presents strategies inspired from high-throughput screening techniques implemented in drug discovery to exploit the potential of a growing CAP library. These strategies are expected to promote the use of a broader spectrum of CAPs, which in turn could lead to improved cell culture models, implants, and wound dressings.

Hydrogels have been developed as extracellular matrix (ECM) mimics both for therapeutic applications and basic biological studies. In particular, elastin-like polypeptide (ELP) hydrogels, which can be tuned to mimic several biochemical and physical characteristics of native ECM, have been constructed to encapsulate various types of cells to create in vitro mimics of in vivo tissues. However, ELP hydrogels become opaque at body temperature because of ELP's lower critical solution temperature behavior. This opacity obstructs light-based observation of the morphology and behavior of encapsulated cells. In order to improve the transparency of ELP hydrogels for better imaging, we have designed a hybrid ELP-polyethylene glycol (PEG) hydrogel system that rapidly cross-links with tris(hydroxymethyl) phosphine (THP) in aqueous solution via Mannich-type condensation. As expected, addition of the hydrophilic PEG component significantly improves the light transmittance. Coherent anti-Stokes Raman scattering (CARS) microscopy reveals that the hybrid ELP-PEG hydrogels have smaller hydrophobic ELP aggregates at 37 degrees C. Importantly, this hydrogel platform enables independent tuning of adhesion ligand density and matrix stiffness, which is desirable for studies of cell-matrix interactions. Human fibroblasts encapsulated in these hydrogels show high viability (>98%) after 7 days of culture. High-resolution confocal microscopy of encapsulated fibroblasts reveals that the cells adopt a more spread morphology in response to higher RGD ligand concentrations and softer gel mechanics.

Designing versatile functional medical adhesives with injectability, self-healing, and strong adhesion is of great significance to achieve desirable therapeutic effects for promoting wound sealing in healthcare. Herein, a self-healing injectable adhesive is fabricated by physical interaction of polyphenol compound tannic acid (TA) and eight-arm poly(ethylene glycol) end-capped with succinimide glutarate active ester (PEG-SG). The hydrogen bonding induced from the structural unit (-CH2-CH2-O-) of PEG and catechol hydroxyl (-OH) of TA, accompanied by ester exchange between N-hydroxysuccinimide (-NHS) and amino (-NH2) of proteins, contributes to self-healing ability and rapid strong adhesion. Notably, the PEG/TA adhesive can repeatedly adhere to rigid porcine tissues, close the coronary artery under a large incision tension, and bear a heavy load of 2 kg. By exhibiting shear-thinning and anti-swelling properties, the PEG/TA adhesive can be easily applied through single-syringe extrusion onto various wounds. The single-channel toothpaste-like feature of the adhesive ensures its storage hermetically for portable usage. Moreover, in vivo operation and histological H&E staining results indicate that the PEG/TA adhesive greatly accelerates wound healing and tissue regeneration in a rat model. With the specialty of injectability, instant self-healing, and long-lasting strong adhesion to facilitate excellent therapeutic effects, the multifunctional PEG/TA adhesive may provide a new alternative for self-rescue and surgical situations.

Escalating cases of organ shortage and donor scarcity worldwide are alarming reminders of the need for alternatives to allograft tissues. Within the last three decades, research efforts in the field of regenerative medicine and tissue engineering continue to address the unmet need for artificial tissues and organs for transplant. Work in the field has evolved to create what we consider a new field, Regenerative Engineering, defined as the Convergence of advanced materials science, stem cell science, physics, developmental biology and clinical translation towards the regeneration of complex tissues and organ systems. Included in the regenerative engineering paradigm is advanced manufacturing. Three-dimensional (3D) bioprinting is a promising and innovative biofabrication strategy to precisely position biologics, including living cells and extracellular matrix (ECM) components, in the prescribed 3D hierarchal organization to create artificial multi-cellular tissues/organs. In this review, we outline recent progress in several bioprinting technologies used to engineer scaffolds with requisite mechanical, structural, and biological complexity. We examine the process parameters affecting bioprinting and bioink-biomaterials and review notable studies on bioprinted skin, cardiac, bone, cartilage, liver, lung, neural, and pancreatic tissue. We also focus on other 3D bioprinting application areas including cancer research, drug testing, high-throughput screening (HTS), and organ-on-a-chip models. We also highlight the current challenges associated with the clinical translation of 3D bioprinting and conclude with the future perspective of bioprinting technology.

3D printing has triggered the acceleration of numerous research areas in health sciences, which traditionally used cells as starting materials, in particular tissue engineering, regenerative medicine and also in the design of more relevant bioassays for drug discovery and development. While cells can be successfully printed in 2D layers without the help of any supporting biomaterial, the obtainment of more complex 3D architectures requires a specific bioink, i.e. a material in which the cells are embedded during and after the printing process helping to support them while they are arranged in superimposed layers. The bioink plays a critical role in bioprinting: first, it must be adapted to the 3D printing technology; then, it must fulfil the physicochemical and mechanical characteristics of the target construct (e.g. stiffness, elasticity, robustness, transparency); finally it should guarantee cell viability and eventually induce a desired behaviour. This review focuses on the nature of bioink components of natural or synthetic origin, and highlights the chemistry required for the establishment of the 3D network in conditions compatible with the selected 3D printing technique and cell survival.

Biomaterials have been used for a long time in the field of medicine. Since the success of

3D printing/bioprinting are promising techniques to fabricate scaffolds with well controlled and patient-specific structures and architectures for bone tissue engineering. In this study, we developed a composite bioink consisting of silk fibroin (SF), gelatin (GEL), hyaluronic acid (HA), and tricalcium phosphate (TCP) and 3D bioprinted the silk fibroin-based hybrid scaffolds. The 3D bioprinted scaffolds with dual crosslinking were further treated with human platelet-rich plasma (PRP) to generate PRP coated scaffolds. Live/Dead and MTT assays demonstrated that PRP treatment could obviously promote the cell growth and proliferation of human adipose derived mesenchymal stem cells (HADMSC). In addition, the treatment of PRP did not significantly affect alkaline phosphatase (ALP) activity and expression, but significantly upregulated the gene expression levels of late osteogenic markers. This study demonstrated that the 3D printing of silk fibroin-based hybrid scaffolds, in combination with PRP post-treatment, might be a more efficient strategy to promote osteogenic differentiation of adult stem cells and has significant potential to be used for bone tissue engineering.

Bioprinting is an emerging approach for fabricating cell-laden 3D scaffolds via robotic deposition of cells and biomaterials into custom shapes and patterns to replicate complex tissue architectures. Bioprinting uses hydrogel solutions called bioinks as both cell carriers and structural components, requiring bioinks to be highly printable while providing a robust and cell-friendly microenvironment. Unfortunately, conventional hydrogel bioinks have not been able to meet these requirements and are mechanically weak due to their heterogeneously crosslinked networks and lack of energy dissipation mechanisms. Advanced bioink designs using various methods of dissipating mechanical energy are aimed at developing next-generation cellularized 3D scaffolds to mimic anatomical size, tissue architecture, and tissue-specific functions. These next-generation bioinks need to have high print fidelity and should provide a biocompatible microenvironment along with improved mechanical properties. To design these advanced bioink formulations, it is important to understand the structure-property-function relationships of hydrogel networks. By specifically leveraging biophysical and biochemical characteristics of hydrogel networks, high performance bioinks can be designed to control and direct cell functions. In this review article, current and emerging approaches in hydrogel design and bioink reinforcement techniques are critically evaluated. This bottom-up perspective provides a materials-centric approach to bioink design for 3D bioprinting.

Biomaterials based upon elastin and elastin-derived molecules are increasingly investigated for their application in tissue engineering. This interest is fuelled by the remarkable properties of this structural protein, such as elasticity, self-assembly, long-term stability, and biological activity. Elastin can be applied in biomaterials in various forms, including insoluble elastin fibres, hydrolysed soluble elastin, recombinant tropoelastin (fragments), repeats of synthetic peptide sequences and as block copolymers of elastin, possibly in combination with other (bio)polymers. In this review, the properties of various elastin-based materials will be discussed, and their current and future applications evaluated.

Recombinant protein-polymer scaffolds such as elastin-like polypeptides (ELPs) offer drug-delivery opportunities including biocompatibility, monodispersity, and multifunctionality. We recently reported that the fusion of FK-506 binding protein 12 (FKBP) to an ELP nanoparticle (FSI) increases rapamycin (Rapa) solubility, suppresses tumor growth in breast cancer xenografts, and reduces side effects observed with free-drug controls. This new report significantly advances this carrier strategy by demonstrating the coassembly of two different ELP diblock copolymers containing drug-loading and tumor-targeting domains. A new ELP nanoparticle (ISR) was synthesized that includes the canonical integrin-targeting ligand (Arg-Gly-Asp, RGD). FSI and ISR mixed in a 1:1 molar ratio coassemble into bifunctional nanoparticles containing both the FKBP domain for Rapa loading and the RGD ligand for integrin binding. Coassembled nanoparticles were evaluated for bifunctionality by performing in vitro cell-binding and drug-retention assays and in vivo MDA-MB-468 breast tumor regression and tumor-accumulation studies. The bifunctional nanoparticle demonstrated superior cell target binding and similar drug retention to FSI; however, it enhanced the formulation potency, such that tumor growth was suppressed at a 3-fold lower dose compared to an untargeted FSI-Rapa control. This data suggests that ELP-mediated scaffolds are useful tools for generating multifunctional nanomedicines with potential activity in cancer.

The linker between the targeting moiety and the nanoparticle is often overlooked when engineering targeted drug delivery vehicles. We hypothesized that pH-triggered conformational changes of an elastin-like peptide (ELP) linker, with repeating VPGVG sequences, could alter the binding affinity of the well-established targeting moiety arginine-glycine-aspartic acid (RGD), which is known to enhance the delivery of nanoparticles to tumor cells via integrin overexpression. The pH change from blood (pH 7.4) to the tumor environment (pH 6) was used to elicit a conformational change in the ELP linker, as described by circular dichroism. Atomic force microscopy confirmed that RGD-ELP resulted in stronger adhesion to both MDA-MB-231 and HCC1806 breast cancer cells at pH 6 relative to pH 7.4. No change in adhesion force was measured as a function of pH for the non-neoplastic MCF-10A cell line and the nontargeting GDR-ELP peptide. This translated to significant binding and uptake of RGD-ELP modified liposomes at pH 6.0 relative to pH 7.4. These results indicate that the pH-triggered conformational structure of the ELP linker shifts RGD-mediated cancer cell targeting from non-active (pH 7.4) to active (pH 6). The reversible shift in ELP secondary structure may be used to engineer targeted drug delivery vehicles with tunable uptake.