Silk Fibroin Microneedles for Transdermal Vaccine Delivery.

Microneedles represent an exciting departure from the existing parenteral injection paradigm for drug delivery, particularly for the administration of vaccines. While the benefit of delivering vaccine antigens to immunocompetent tissue in the skin has been established, there have been varying degrees of success using microneedles to do so. Here, we investigate the use of silk fibroin protein to produce microneedles and evaluate their ability to deliver vaccines against influenza, Clostridium difficile, and Shigella. Fibroin protein from the silkworm Bombyx mori possesses suitable properties for use in a microneedle system, including all-aqueous processing, mechanical strength in dried formats, biocompatibility, and the ability to temperature stabilize biomacromolecules. As such, this biomaterial combines the processing and biocompatibility advantages of a dissolving microneedle system with the product stability and mechanical strength of coated insoluble microneedles. Through successful vaccination of mice against three separate antigens, we establish that silk fibroin is well-suited for use as a solid-coated microneedle delivery system and offers long-term potential similar to that of the leading microneedle biomaterials.

In vitro chondrogenesis with lysozyme susceptible bacterial cellulose as a scaffold.

A current focus of tissue engineering is the use of adult human mesenchymal stem cells (hMSCs) as an alternative to autologous chondrocytes for cartilage repair. Several natural and synthetic polymers (including cellulose) have been explored as a biomaterial scaffold for cartilage tissue engineering. While bacterial cellulose (BC) has been used in tissue engineering, its lack of degradability in vivo and high crystallinity restricts widespread applications in the field. Recently we reported the formation of a novel bacterial cellulose that is lysozyme-susceptible and -degradable in vivo from metabolically engineered Gluconacetobacter xylinus. Here we report the use of this modified bacterial cellulose (MBC) for cartilage tissue engineering using hMSCs. MBC's glucosaminoglycan-like chemistry, combined with in vivo degradability, suggested opportunities to exploit this novel polymer in cartilage tissue engineering. We have observed that, like BC, MBC scaffolds support cell attachment and proliferation. Chondrogenesis of hMSCs in the MBC scaffolds was demonstrated by real-time RT-PCR analysis for cartilage-specific extracellular matrix (ECM) markers (collagen type II, aggrecan and SOX9) as well as histological and immunohistochemical evaluations of cartilage-specific ECM markers. Further, the attachment, proliferation, and differentiation of hMSCs in MBC showed unique characteristics. For example, after 4 weeks of cultivation, the spatial cell arrangement and collagen type-II and ACAN distribution resembled those in native articular cartilage tissue, suggesting promise for these novel in vivo degradable scaffolds for chondrogenesis.

A cellulosic responsive "living" membrane.

Bacterial cellulose has been demonstrated to be a remarkably versatile biomaterial and widely used in biomedical applications due to its unique physical properties. Here we reported for the first time a "living membrane" system based on recombinant Escherichia coli bacterial strains entrapped in cellulosic membranes produced by Gluconacetobacter xylinus. Biologically driven detection and identification of a range of target molecules presents unique challenges, and requires that detection methods are developed to be rapid, specific and sensitive. The compatibility of G. xylinus and recombinant E. coli strains was first investigated for co-cultivation, and the relationship between the number of entrapped E. coli and the level of inducible signal achieved was further explored by fluorescent signal observation in confocal microscopy. Finally to amplify the response to inducers for maximum fluorescent signal, a positive-feedback genetic amplifier was designed within recombinant E. coli strain entrapped in the living cellulosic membrane system, allowing for the detection mechanism to be extremely sensitive and resulting in a significant fluorescent signal from a single receptor binding event. The living membrane system proposed here will create devices of greater complexity in function for applications in biological and chemical detection.

Antibiotic-Releasing Silk Biomaterials for Infection Prevention and Treatment.

Effective treatment of infections in avascular and necrotic tissues can be challenging due to limited penetration into the target tissue and systemic toxicities. Controlled release polymer implants have the potential to achieve the high local concentrations needed while also minimizing systemic exposure. Silk biomaterials possess unique characteristics for antibiotic delivery including biocompatibility, tunable biodegradation, stabilizing effects, water-based processing and diverse material formats. We report on functional release of antibiotics spanning a range of chemical properties from different material formats of silk (films, microspheres, hydrogels, coatings). The release of penicillin and ampicillin from bulk-loaded silk films, drug-loaded silk microspheres suspended in silk hydrogels and bulk-loaded silk hydrogels was investigated and in vivo efficacy of ampicillin-releasing silk hydrogels was demonstrated in a murine infected wound model. Silk sponges with nanofilm coatings were loaded with gentamicin and cefazolin and release was sustained for 5 and 3 days, respectively. The capability of silk antibiotic carriers to sequester, stabilize and then release bioactive antibiotics represents a major advantage over implants and pumps based on liquid drug reservoirs where instability at room or body temperature is limiting. The present studies demonstrate that silk biomaterials represent a novel, customizable antibiotic platform for focal delivery of antibiotics using a range of material formats (injectable to implantable).

Sustained volume retention in vivo with adipocyte and lipoaspirate seeded silk scaffolds.

Current approaches to soft tissue regeneration include the use of fat grafts, natural or synthetic biomaterials as filler materials. Fat grafts and natural biomaterials resorb too quickly to maintain tissue regeneration, while synthetic materials do not degrade or regenerate tissue. Here, we present a simple approach to volume stable filling of soft tissue defects. In this study, we combined lipoaspirate with a silk protein matrix in a subcutaneous rat model. Silk biomaterials can be tailored to fit a variety of needs, and here were processed silk biomaterials into a porous sponge format to allow for tissue ingrowth while remaining mechanically robust. Over an 18 month period, the lipoaspirate seeded silk protein matrix regenerated subcutaneous adipose tissue while maintaining the original implanted volume. A silk protein matrix alone was not sufficient to regenerate adipose tissue, but yielded a fibrous tissue, although implanted volume was maintained. This work presents a significant improvement to the standard approaches to filling soft tissue defects by matching biomaterial degradation and tissue regeneration profiles.

Functionalized silk biomaterials for wound healing.

Silk protein-biomaterial wound dressings with epidermal growth factor (EGF) and silver sulfadiazine were studied with a cutaneous excisional mouse wound model. Three different material designs and two different drug incorporation techniques were studied to compare wound healing responses. Material formats included silk films, lamellar porous silk films and electrospun silk nanofibers, each studied with the silk matrix alone and with drug loading or drug coatings on the silk matrices. Changes in wound size and histological assessments of wound tissues showed that the functionalized silk biomaterial wound dressings increased wound healing rate, including reepithelialization, dermis proliferation, collagen synthesis and reduced scar formation, when compared to air-permeable Tegaderm tape (3M) (- control) and a commercial wound dressing, Tegaderm Hydrocolloid dressing (3M) (+ control). All silk biomaterials were effective for wound healing, while the lamellar porous films and electrospun nanofibers and the incorporation of EGF/silver sulfadiazine, via drug loading or coating, provided the most rapid wound healing responses. This systematic approach to evaluating functionalized silk biomaterial wound dressings demonstrates a useful strategy to select formulations for further study towards new treatment options for chronic wounds.

Laminar silk scaffolds for aligned tissue fabrication.

3D-biomaterial scaffolds with aligned architecture are of vital importance in tissue regeneration. A generic method is demonstrated to produce aligned biomaterial scaffolds using the physics of directional ice freezing. Homogeneously aligned 3D silk scaffolds with high porosity and alignment are prepared. The method can be adapted to a wide range of polymers and is devoid of any chemical reactions, thus avoiding potential complications associated with by-products. Mechanical properties and cellular responses with chondrocytes and bone-marrow-derived hMSCs are studied, assessing survival, proliferation, and differentiation. In vivo tests suggest biocompatibility of the matrices for future tissue engineering applications, specifically in areas where high cellular alignment is needed.

Implantable, multifunctional, bioresorbable optics.

Advances in personalized medicine are symbiotic with the development of novel technologies for biomedical devices. We present an approach that combines enhanced imaging of malignancies, therapeutics, and feedback about therapeutics in a single implantable, biocompatible, and resorbable device. This confluence of form and function is accomplished by capitalizing on the unique properties of silk proteins as a mechanically robust, biocompatible, optically clear biomaterial matrix that can house, stabilize, and retain the function of therapeutic components. By developing a form of high-quality microstructured optical elements, improved imaging of malignancies and of treatment monitoring can be achieved. The results demonstrate a unique family of devices for in vitro and in vivo use that provide functional biomaterials with built-in optical signal and contrast enhancement, demonstrated here with simultaneous drug delivery and feedback about drug delivery with no adverse biological effects, all while slowly degrading to regenerate native tissue.

A physically transient form of silicon electronics.

A remarkable feature of modern silicon electronics is its ability to remain physically invariant, almost indefinitely for practical purposes. Although this characteristic is a hallmark of applications of integrated circuits that exist today, there might be opportunities for systems that offer the opposite behavior, such as implantable devices that function for medically useful time frames but then completely disappear via resorption by the body. We report a set of materials, manufacturing schemes, device components, and theoretical design tools for a silicon-based complementary metal oxide semiconductor (CMOS) technology that has this type of transient behavior, together with integrated sensors, actuators, power supply systems, and wireless control strategies. An implantable transient device that acts as a programmable nonantibiotic bacteriocide provides a system-level example.

Stabilization of vaccines and antibiotics in silk and eliminating the cold chain.

Sensitive biological compounds, such as vaccines and antibiotics, traditionally require a time-dependent "cold chain" to maximize therapeutic activity. This flawed process results in billions of dollars worth of viable drug loss during shipping and storage, and severely limits distribution to developing nations with limited infrastructure. To address these major limitations, we demonstrate self-standing silk protein biomaterial matrices capable of stabilizing labile vaccines and antibiotics, even at temperatures up to 60 °C over more than 6 months. Initial insight into the mechanistic basis for these findings is provided. Importantly, these findings suggest a transformative approach to the cold chain to revolutionize the way many labile therapeutic drugs are stored and utilized throughout the world.

Materials and designs for wirelessly powered implantable light-emitting systems.

Strategies are presented to achieve bendable and stretchable systems of microscale inorganic light-emitting diodes with wireless powering schemes, suitable for use in implantable devices. The results include materials strategies, together with studies of the mechanical, electronic, thermal and radio frequency behaviors both in vitro and in in-vivo animal experiments.

High-strength silk protein scaffolds for bone repair.

Biomaterials for bone tissue regeneration represent a major focus of orthopedic research. However, only a handful of polymeric biomaterials are utilized today because of their failure to address critical issues like compressive strength for load-bearing bone grafts. In this study development of a high compressive strength (~13 MPa hydrated state) polymeric bone composite materials is reported, based on silk protein-protein interfacial bonding. Micron-sized silk fibers (10-600 µm) obtained utilizing alkali hydrolysis were used as reinforcement in a compact fiber composite with tunable compressive strength, surface roughness, and porosity based on the fiber length included. A combination of surface roughness, porosity, and scaffold stiffness favored human bone marrow-derived mesenchymal stem cell differentiation toward bone-like tissue in vitro based on biochemical and gene expression for bone markers. Further, minimal in vivo immunomodulatory responses suggested compatibility of the fabricated silk-fiber-reinforced composite matrices for bone engineering applications.

Focal Infection Treatment using Laser-Mediated Heating of Injectable Silk Hydrogels with Gold Nanoparticles.

Medical treatment of subcutaneous bacterial abscesses usually involves systemic high-dose antibiotics and incision-drainage of the wound. Such an approach suffers from two main deficiencies: bacterial resistance to antibiotics and pain associated with multiple incision-drainage-wound packing procedures. Furthermore, the efficacy of high-dose systemic antibiotics is limited because of the inability to penetrate into the abscess. To address these obstacles, we present a treatment relying on laser-induced heating of gold nanoparticles embedded in an injectable silk-protein hydrogel. Although bactericidal nanoparticle systems have been previously employed based on silver and nitric oxide, they have limitations regarding customization and safety. The method we propose is safe and uses biocompatible, highly tunable materials: an injectable silk hydrogel and Au nanoparticles, which are effective absorbers at low laser powers such as those provided by hand held devices. We demonstrate that a single 10-minute laser treatment of a subcutaneous infection in mice preserves the general tissue architecture, while achieving a bactericidal effect - even resulting in complete eradication in some cases. The unique materials platform presented here can provide the basis for an alternative treatment of focal infections.

N-acetylglucosamine 6-phosphate deacetylase (nagA) is required for N-acetyl glucosamine assimilation in Gluconacetobacter xylinus.

Metabolic pathways for amino sugars (N-acetylglucosamine; GlcNAc and glucosamine; Gln) are essential and remain largely conserved in all three kingdoms of life, i.e., microbes, plants and animals. Upon uptake, in the cytoplasm these amino sugars undergo phosphorylation by phosphokinases and subsequently deacetylation by the enzyme N-acetylglucosamine 6-phosphate deacetylase (nagA) to yield glucosamine-6-phosphate and acetate, the first committed step for both GlcNAc assimilation and amino-sugar-nucleotides biosynthesis. Here we report the cloning of a DNA fragment encoding a partial nagA gene and its implications with regard to amino sugar metabolism in the cellulose producing bacterium Glucoacetobacter xylinus (formally known as Acetobacter xylinum). For this purpose, nagA was disrupted by inserting tetracycline resistant gene (nagA::tet(r); named as ΔnagA) via homologous recombination. When compared to glucose fed conditions, the UDP-GlcNAc synthesis and bacterial growth (due to lack of GlcNAc utilization) was completely inhibited in nagA mutants. Interestingly, that inhibition occured without compromising cellulose production efficiency and its molecular composition under GlcNAc fed conditions. We conclude that nagA plays an essential role for GlcNAc assimilation by G. xylinus thus is required for the growth and survival for the bacterium in presence of GlcNAc as carbon source. Additionally, G. xylinus appears to possess the same molecular machinery for UDP-GlcNAc biosynthesis from GlcNAc precursors as other related bacterial species.

Silk fibroin and polyethylene glycol-based biocompatible tissue adhesives.

Tissue sealants have emerged in recent years as strong candidates for hemostasis. A variety of formulations are currently commercially available and though they satisfy many of the markets' needs there are still key aspects of each that need improvement. Here we present a new class of blends, based on silk fibroin and chemically active polyethylene glycols (PEGs) with strong adhesive properties. These materials are cytocompatible, crosslink within seconds via chemical reaction between thiols and maleimides present on the constituent PEGs and have the potential to further stabilize through ß-sheet formation by silk. Based on the silk concentration in the final formulation, the adhesive properties of these materials are comparable or better than the current leading PEG-based sealant. In addition, the silk-PEG based materials show decreased swelling and longer degradation times. Such properties would make them suitable for applications for which the current sealants are contraindicated.

Waterproof AlInGaP optoelectronics on stretchable substrates with applications in biomedicine and robotics.

Inorganic light-emitting diodes and photodetectors represent important, established technologies for solid-state lighting, digital imaging and many other applications. Eliminating mechanical and geometrical design constraints imposed by the supporting semiconductor wafers can enable alternative uses in areas such as biomedicine and robotics. Here we describe systems that consist of arrays of interconnected, ultrathin inorganic light-emitting diodes and photodetectors configured in mechanically optimized layouts on unusual substrates. Light-emitting sutures, implantable sheets and illuminated plasmonic crystals that are compatible with complete immersion in biofluids illustrate the suitability of these technologies for use in biomedicine. Waterproof optical-proximity-sensor tapes capable of conformal integration on curved surfaces of gloves and thin, refractive-index monitors wrapped on tubing for intravenous delivery systems demonstrate possibilities in robotics and clinical medicine. These and related systems may create important, unconventional opportunities for optoelectronic devices.

Dissolvable films of silk fibroin for ultrathin conformal bio-integrated electronics.

Electronics that are capable of intimate, non-invasive integration with the soft, curvilinear surfaces of biological tissues offer important opportunities for diagnosing and treating disease and for improving brain/machine interfaces. This article describes a material strategy for a type of bio-interfaced system that relies on ultrathin electronics supported by bioresorbable substrates of silk fibroin. Mounting such devices on tissue and then allowing the silk to dissolve and resorb initiates a spontaneous, conformal wrapping process driven by capillary forces at the biotic/abiotic interface. Specialized mesh designs and ultrathin forms for the electronics ensure minimal stresses on the tissue and highly conformal coverage, even for complex curvilinear surfaces, as confirmed by experimental and theoretical studies. In vivo, neural mapping experiments on feline animal models illustrate one mode of use for this class of technology. These concepts provide new capabilities for implantable and surgical devices.

Emulsan-alginate beads for protein adsorption.

Emulsan-alginate beads were prepared and challenged using bovine serum albumin (BSA) to assess adsorption in comparison to alginate beads. BSA binding to the emulsan-alginate beads was improved over the alginate bead controls and protein adsorption was less sensitive to changes in ionic strength. BSA adsorption between pH 8.5 and 5.3 in alginate beads was 2-3-times lower compared to the emulsan-alginate beads in the same pH range. BSA adsorption and kinetic constants were at least 2-times higher for the emulsan-alginate beads compared to the alginate controls based on the Langmuir adsorption model. To further explore the utility of these novel emulsan-alginate bead systems, complex cell-free supernatants from some pathogenic microorganisms were exposed to the emulsan-alginate beads and increased protein adsorption was found when compared to controls. These trends were also confirmed with alpha-hemolysin toxicity studies. The data suggest that the protein-binding capacity of emulsan-alginate beads exceeds alginate controls, attributable to the unique binding features of emulsan.

Erratum: "Silicon electronics on silk as a path to bioresorbable, implantable devices" [Appl. Phys. Lett. 95, 133701 (2009)].

[This corrects the article on p. 133701 in vol. 95.].