The authors thank Rowan University for support of this research. XH also thanks Dr. David L. Kaplan at Tufts University and the NIH P41 Tissue Engineering Resource Center (TERC) for previous technical trainings.&#160;

No conflicts of interest declared.

One of the most critical procedures in producing &#8220;alloy&#8221; protein system is to verify the miscibility of the blended proteins. Otherwise, it is only an immiscible protein mixture or protein composite system without stable and tunable properties. An experimental thermal analysis method can be used for this purpose and to confirm their alloy properties. Protein-protein interactions can be viewed according to Flory-Huggins&#8217;s lattice model48 as interactions between the &#8220;solvent&#8221; (the predominant protein component) and the &#8220;solute&#8221; (the minor protein component). Based on this model, the free energy change during mixing between the &#8220;solvent&#8221; and the &#8220;solute&#8221; governs the miscibility of the blend48. Generally, there are three different degrees of miscibility: (a) fully miscible (form one-phase material macroscopically), (b) metastable (form semi-miscible phases in the material), and (c) immiscible (remain original two-phases with individual protein A and B domains)3,48. Correspondingly, the glass transition behaviors of a two protein system can demonstrate these differences and ideally can be expressed using a phase diagram model (Figure&#160;6). For example, if protein A and B have their individual single glass transition temperatures, Tg(A) and Tg(B), respectively (Figure&#160;6 green and blue curves), a fully miscible protein alloy system should show only one glass transition during the heating. This single glass transition is normally intermediate between the Tgs of A and B (Figure&#160;6). Otherwise, the proteins could form an immiscible blend whereby both Tg(A) and Tg(B) appear at their original positions (Figure&#160;6). The proteins may also form a semi-miscible system indicated by two shifted glass transitions (Figure&#160;6). A practical example of fully miscible protein &#8220;alloy&#8221; system (form one-glass transition) can be found in the DSC scans of silk-tropoelastin blend samples in Figure 4 (thermal application)9. With the decrease of silk content, the glass transition temperatures (Tg) of the blends increased gradually from 178 &#176;C&#160;(pure silk) to 190 &#176;C (pure tropoelastin)9, yet consistently keep a homogeneous single glass transition for all type of blends. According to Flory-Huggins&#8217;s model, this indicates that all silk-tropoelastin blends of various mixing ratios are stable and are fully miscible protein alloys without any macrophase separations.
In conclusion, new generations of protein alloy materials can be produced and fabricated into different medical devices (e.g., films, sutures, screws, plates, micro-needles, gels), with controlled and tunable optical, electrical, chemical, thermal, and mechanical properties. Through controlling the mixing components and ratios, various biophysical properties of protein alloy materials, such as elasticity, strength, surface roughness, surface charge, biodurability and chemical activity, can be manipulated, which could ultimately impact the tissue functions as well as the local cellular behaviors associated with these materials. Additionally, due to the nature of proteins&#8217; programmable lifetime, in vivo becomes a viable platform for these alloy materials. Such advantages could provide novel options for implantable medical devices in the future where post repair surgical retrieval can be avoided. These protein alloy biomaterials would also offer a new pathway to produce medical devices with tunable biological functions and properties, and with matching tissue compliance and related needs. This article provides a general protocol review for how to fabricate these devices and would benefit both scientists and clinical doctors in multiple biomedical fields.

Nature has created strategies to generate tunable and multifunctional biological matrixes using a limited number of structural proteins. For example, elastins and collagens are always used together in vivo to provide the adjustable strengths and functions required for specific tissues1,2. The key to this strategy is the blending. Blending involves mixing proteins with specific ratios and is a technological approach to generate simple material systems with tunable and varied properties3-5. Compared with synthetic engineering strategies6,7, blending can also improve material uniformity and the ability to process the material due to the ease of operation8-16. Therefore, designing multifunctional, biocompatible protein alloy materials is an emerging area of medical research. This technology will also provide systematic knowledge of the impact of natural protein matrices on cell and tissue functions both in vitro and in vivo10,17. By optimizing molecular interfaces between different proteins, protein-based alloy materials can encompass a range of physical functions, such as thermal stability at different temperatures, elasticity to support diverse tissues, electrical sensitivity in variable organs, and optical properties for corneal tissue regeneration3,18-27. The outcome of these studies will provide a new protein-materials platform in the field of biomedical science with direct relevance to tunable tissue repairs and disease treatments and further lead to biodegradable implant devices where their novel therapeutic and diagnostic features can be envisioned3.
Many natural structural proteins have critical physical and bioactive properties that can be exploited as candidates for the biomaterial matrixes. Silks from different worm species, keratins from hairs and wools, elastins and collagens from different tissues, and various plant proteins are some of the most common structural proteins used for designing variable protein-based materials (Figure 1)18-27. In general, these proteins can form different molecular secondary structures (e.g., beta sheets for silks, or coiled coils for keratins) due to their unique repetitive primary amino acid sequences3,28-35. These features promote the formation of self-assembled macroscopic structures with unique functions at biological interfaces prompting their utility as a treasured resource of biopolymer materials. Here, two types of structural proteins were used (protein A from wild tussah silk and protein B from domesticated mulberry silk as an example) to demonstrate the general protocols of producing various protein alloy biomaterials. The protocols demonstrated include part 1: protein interaction predictions and simulations, part 2: production of protein alloy solutions, and part 3: fabrication of protein alloy systems and for optical, electrical, and pharmaceutical applications.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/50891/50891fig1highres.jpg" width="500" /> 
Figure 1. Raw materials of various structural proteins that are commonly used in our laboratory for designing protein-based materials, including silks from different worm species, keratins from hairs and wools, elastins from different tissues, and various plant proteins.

<table border="0" cellpadding="0" cellspacing="0" width="1050">
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Q100 Differential Scanning Calorimeters (DSC)</td>
			<td style="width:143px;">TA Instruments, New Castle, DE, USA 
			&#160;</td>
			<td style="width:169px;">N/A</td>
			<td style="width:523px;">You can use any type of DSC with a software to calculate the heat capacity</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">SS30T Vacuum Sputtering System&#160;</td>
			<td style="width:143px;">T-M Vacuum Products, Inc., Cinnaminson, NJ, USA</td>
			<td style="width:169px;">N/A</td>
			<td>With custom built parts; You can use any type of sputtering system to coat</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">VWR 1415M Vacuum Oven&#160;</td>
			<td style="width:143px;">VWR International, Bridgeport, NJ, USA</td>
			<td style="width:169px;">N/A</td>
			<td>You can use any type of vacuum oven to physically crosslink the samples</td>
		</tr>
	</tbody>
</table>

designing silk-silk protein alloy materials for biomedical applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Typical protein-protein interactions (e.g., between protein A and protein B) could contain charge-charge (electrostatic) attractions, hydrogen bonding formation, hydrophobic-hydrophilic interactions, as well as dipole, solvent, counter ion, and entropic effects between the specific domains of the two proteins (Figure 2)3. Therefore, fundamentally, we can predict the effects of these interactions by computational simulations.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/50891/50891fig2highres.jpg" width="500" /> 
Figure 2. Interactions between protein A and protein B. Typically, these interactions could be based on charge-charge (electrostatic) attractions, hydrogen bonding formation, or hydrophobic-hydrophilic interactions between the specific domains of these two proteins.
The protein-alloy system can be modeled as a material composed of crosslinked protein sub-alloy domains, where each of these sub-alloys is assumed to be stable. The interactions between the proteins (A and B) can be considered as bonds with different stiffnesses (for this study, we consider only two types of weak or strong bonds in Figure 3). The weak bonds could represent hydrogen bonds and other bonds described in Figure 2. The protein-alloy as a whole is formed through dual cross-linkages between many sub-alloys that bounded together through both strong and weak bonds. The sub-alloys are formed using strong bonds and within the sub-alloys we allow the formation of weaker bonds. The protein-alloy as a whole is formed through dual cross-linkages between many sub-alloys that bound together through various strong and weak bonds. When the system is sufficiently stressed, both the weak and the strong bonds are ruptured. Under right conditions the weak bonds are allowed to reform the linkages again. However, the stronger bonds will be ruptured irreversibly. The existence of the weak bonds allows the alloy-protein to keep its structural integrity under external stress36-41. This numerical simulation uses a mathematical model developed through a bottom-up approach that is based on finite element methods through lattice-spring model36-41.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/50891/50891fig3highres.jpg" width="500" /> 
Figure 3. Computational simulation to demonstrate the mechanical advantage of a protein alloy system during stretching. During the stretching simulation, one type of protein (blue color) can form an elastic network like springs providing super-elasticity for the materials, while another type of protein (green color) can provide strong physical crosslinkers for stabilizing the material network. Dynamic structural transitions (e.g., hydrogen bond formations and deformations) could be induced in different domains for storing and releasing energy or providing additional mechanical support during the stretching.
Figure 3 demonstrates a typical computational simulation of the mechanical properties of a protein alloy system (with wild tussah silk as protein A and domesticated mulberry silk as protein B) during stretching. During the stretching simulation, one type of protein (blue color) can form an elastic network with springs providing super-elasticity for the materials, while another type of protein (green color) can serve as particles with strong physical crosslinkers for the material network. Dynamic structural transitions (e.g., hydrogen bond formation and deformation) can be induced in different domains for storing and releasing energy or providing additional mechanical support during the stretch. The simulation studies gives a general theoretical picture to understand the interactions between different structural protein molecules, such that useful pairs of proteins could be picked to generate protein alloy materials with strong interactions and specific properties, such as extraordinary mechanical elasticity.
Generally, once the protein A and B are selected (here they are wild silk and domesticated silk), a protein alloy solution can be produced in several steps (Figure 4). First, the protein sources like natural fibers or powders should be cleaned or purified. For example, a degumming process could be used to remove the soluble silk sericin proteins coated on most silk fibroin fibers20. Second, a suitable solvent needs to be found to dissolve the insoluble protein sources into solutions. For instance, high concentration LiBr solution is a good solvent to cut beta-sheet secondary structures in different silks and dissolve the fibers into solutions. Third, a dialysis method can be used to remove the dissolving solvent and recover the protein molecules in an aqueous solution. Additional centrifuging is often necessary to remove the impurities and undissolved aggregates in the solution. Finally, different protein aqueous solutions can be mixed together gently with various ratios. Therefore, if the two protein solutions do not have macrophase separation, they will be blended together with strong interactions and form new protein alloy system for different biomedical applications. To make the protein alloy materials insoluble in the body, different physical or chemical crosslinking treatments can be adapted. For example, it is found that high-temperature and high-pressure water vapor annealing could incredibly crosslink different silk or elastin materials6,52. While different keratin materials can be crosslinked by their natural disulfide bonds in the protein side chains53.
Once the protein alloy solutions are produced and verified, they can be formed into a wide range of biomaterials with tunable properties, including material matrixes for thermal, mechanical, optical, electrical, chemical, or biomedical applications (Figure 4). In this article, three emerging applications for these materials to demonstrate the unique advantage of protein alloy biomaterials have been selected (Figure 4). Through modern micro-fabrication techniques, different surface patterns can be generated at micro or nano-scales on protein alloy materials (Figure 4 optical application). For example, if an optical diffraction pattern is produced on the film surface, this film can used as a media to transfer laser beams into different optical pattern22,23. If the protein alloy material was emerged in an enzyme solution, the degradation profile of the films can be understood by comparing the real-time diffraction patterns from the film with the original pattern (instead of back and forth washing and examining the degraded films in the air). Another emerging technique is to coat different micro-scale circuits and wireless resonators on the protein alloy materials (Figure 4 electrical application). Through this technique, micro-currents of damaged tissues or organs in vivo can be monitored, with wireless signals directly transferred to doctors24,51. And the material mechanical toughness and biodurability in the body can be easily controlled by blending ratios and specific protein components of the materials. Finally, different soluble or insoluble cancer drugs can be directly incorporated into the protein alloy materials (Figure 4 chemical application). Cancer drugs are often very toxic, and will damage not only the cancer cells but also the normal human immune system. Therefore, controlling the region and dose of cancer drug delivery per day in the body is one of the most important topics in pharmaceutical science. Through incorporating cancer drugs into protein alloy materials, we can implant the material only into cancer tissues or organs, and control the release rate of the cancer drug per day from protein alloy network by controlling the protein components and mixing ratios. Since the protein matrix is completely biodegradable, the protein alloy materials will be automatically removed by body enzymes after the drug releasing period. The residues of protein alloy materials are purely amino acids, which can be absorbed by the body naturally to further produce other essential proteins in vivo. Patients who get cured by controlled release of cancer drugs from implanted protein alloy materials will finally recovered without additives in the body, and both natural protein alloy matrixes and cancer drugs will be efficiently absorbed in the body during this curing process.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/50891/50891fig4highres.jpg" width="500" /> 
Figure 4. General steps to produce a protein alloy material, including cleaning or purifying the protein material sources, dissolving the insoluble protein materials into solutions, dianalysizing to remove the dissolving solvent from a protein aqueous solution, centrifuging and mixing together with different ratios, and physical or chemical crosslinking treatments. The protein alloy solution can subsequently be formed into a wide range of biomaterials with tunable properties, including material matrixes for thermal, mechanical, optical, electrical, chemical, or biomedical applications. <a href="https://www.jove.com/files/ftp_upload/50891/50891fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Here, we demonstrated the critical procedures of how to fabricate electrical materials on protein alloys with details in Figure 5. Thin metallic films such as electric circuits can be created using several different deposition techniques including evaporation, pulsed laser deposition, or sputtering. Sputtering was selected for the current study since it offers significant flexibility for the energy of the sputtered species through the adjustment of sputtering gas pressure and power applied to the cathode as well as deposition uniformity through adjustment of the gas pressure and cathode size. Sputter deposition can be used to project a metallic film onto a glass substrate (Figure 5A). In this case, Ag circuit films were deposited in a high vacuum chamber with a base pressure of about 1 x 10-7 Torr. The Ar sputtering gas was introduced to the chamber at a pressure of 20 mTorr and the Ag was deposited using an RF generator at 100 W for 20 min from a 2-inch planar magnetron cathode that is 8 cm from the substrate surface. Devices are defined using common photolithographic techniques in the films on glass followed by wet chemical etching (see detailed procedure in Figure 5A). Devices can also be defined by deposition through a physical mask directly onto the protein films. The room temperature electrical resistivity of the electric circuits on the protein films was measured using both two-terminal and four-terminal techniques. The advantage of the four-terminal approach is to eliminate contact resistance from the measurement but we find that the contact resistance is not significant so a two-terminal measurement is sufficient. The two terminal measurements uses a good-quality multimeter set on the ohm scale making contact with the film at both ends of the metal wire (schematically shown in Figure 5B). In this measurement, the multimeter serves as both a current source and a voltmeter and the measured resistance is the voltage divided by the current. The resistivity is calculated using &#961; = RA/l, where R is the resistance, A is the cross-sectional area of the wire and l is the distance between probes. For the created here, the R was measured to be 23.5 &#937; using the slope of voltage-current curve in Figure 5B (R=&#916;Voltage/&#916;Current), l is 4.45 x 10-2 m, and A was found to be 6.685 x 10-10 m2. Using these numbers, a resistivity of 3.6 x 10-7 &#937;&#183;m was found for the films, approximately 20x larger than that for bulk silver metal (1.6 x 10-8 &#937;&#183;m). A higher resistivity measured in films in comparison to bulk is typical due to the already constrained current path and the inability of charge carriers to avoid defects. Heating the metal with a heat gun increased its resistance indicating an increase in the rate of electron scattering by phonons characteristic of metallic conduction.
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/50891/50891fig5highres.jpg" width="500" /> 
Figure 5. (A) Procedure to make electric circuits on protein alloy films (here silver circuits on wild tussah silk and mulberry silk blend films as an example): (a) Uncoated microscope slide; (b) Silver plasma during sputter deposition from a 2 foot diameter target; (c) Silver coated glass slide; (d) Silver coated sample held to the spinner using the vacuum chuck before adding the photoresist; (e) Silver slide coated with photoresist was put into oven for soft-bake at 120 &#176;C; (f) Expose to UV radiation to break the polymer bonds in the photoresist; (g) Develop the photoresist; (h) Hard-bake the resist to prepare for etching in acid; (i) Etch in the acid, then stop the etch by rinsing in a water bath; (j) Dry the slide; (k) Cast protein alloy solution onto the slide; (l) Transfer silver patterns to the dried protein film. (B) A typical voltage-current curve of the produced electric circuits on a protein alloy film. The final electrical resistivity on film is measured around 3.6 &#215; 10-7 &#937;&#183;m, approximately 20x larger than that for bulk silver metal. <a href="https://www.jove.com/files/ftp_upload/50891/50891fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" fo:content-width="5in" src="/files/ftp_upload/50891/50891fig6highres.jpg" width="500" /> 
Figure 6. Thermal analysis model used for verifying miscibility of blended protein system. If protein A and B have individual single glass transition temperatures, Tg(A) and Tg(B), respectively (green and blue curves), a fully miscible protein alloy system will show only one glass transition different from the Tgs of A and B (red curve). Otherwise, the proteins are immiscible mixtures with both Tg(A) and Tg(B) (black curve), or semi-miscible composites with two shifted glass transitions (orange curve).

Watch this Scientific Journal Video about Designing Silk-silk Protein Alloy Materials for Biomedical Applications at JoVE.com

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

designing-silk-silk-protein-alloy-materials-for-biomedical

Merck & Co., Inc.

Research

JoVE Journal

Bioengineering

18.2K Views.  Rowan University. Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

Video: Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Designing a Bio-responsive Robot from DNA Origami

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in parallel computing2,3 , recognize and bind molecular or cellular targets4,5 , catalyze chemical reactions6,7 , and generate calculated responses in a biological system8,9. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures10-12, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.
Creating shapes from nucleic acids was first proposed by Seeman13, and several variations on this theme have since been realized using various techniques11,12,14,15 . However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami16. In this technique, the folding of a long (&gt;7,000 bases) single-stranded DNA 'scaffold' is directed to a desired shape by hundreds of short complementary strands termed 'staples'. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well17,18 .
 
The current paper will focus on the caDNAno 2.0 software19 developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.
In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described20. This robot is 'robotic' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando21 to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here, we describe how DNA origami can be utilized to design a robotic robot capable of sensing biological cues and responding by shape shifting, subsequently relayed to a desired effect.

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in ...

Graphene Coatings for Biomedical Implants

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with nanometer thick layers of graphene for applications as a stent material. Graphene was grown on copper substrates via chemical vapor deposition and then transferred onto nitinol substrates. In order to understand how the graphene coating could change biological response, cell viability of rat aortic endothelial cells and rat aortic smooth muscle cells was investigated. Moreover, the effect of graphene-coatings on cell adhesion and morphology was examined with fluorescent confocal microscopy. Cells were stained for actin and nuclei, and there were noticeable differences between pristine nitinol samples compared to graphene-coated samples. Total actin expression from rat aortic smooth muscle cells was found using western blot. Protein adsorption characteristics, an indicator for potential thrombogenicity, were determined for serum albumin and fibrinogen with gel electrophoresis. Moreover, the transfer of charge from fibrinogen to substrate was deduced using Raman spectroscopy. It was found that graphene coating on nitinol substrates met the functional requirements for a stent material and improved the biological response compared to uncoated nitinol. Thus, graphene-coated nitinol is a viable candidate for a stent material.

Graphene offers potential as a coating material for biomedical implants. In this study we demonstrate a method for coating nitinol alloys with nanometer thick layers of graphene and determine how graphene may influence implant response.

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with ...

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...