Kyle A. Alberti

Pericellular Hydrogel/Nanonets Inhibit Cancer Cells

Fibrils formed by proteins are vital components for cells. However, selective formation of xenogenous nanofibrils of small molecules on mammalian cells has yet to be observed. Here we report an unexpected observation of hydrogel/nanonets of a small D-peptide derivative in pericellular space. Surface and secretory phosphatases dephosphorylate a precursor of a hydrogelator to trigger the self-assembly of the hydrogelator and to result in pericellular hydrogel/nanonets selectively around the cancer cells that overexpress phosphatases. Cell-based assays confirm that the pericellular hydrogel/nanonets block cellular mass exchange to induce apoptosis of cancer cells, including multidrug-resistance (MDR) cancer cells, MES-SA/Dx5. Pericellular hydrogel/nanonets of small molecules to exhibit distinct functions illustrates a fundamentally new way to engineer molecular assemblies spatiotemporally in cellular microenvironment for inhibiting cancer cell growth and even metastasis.

A toolkit of thread-based microfluidics, sensors, and electronics for 3D tissue embedding for medical diagnostics

Threads, traditionally used in the apparel industry, have recently emerged as a promising material for the creation of tissue constructs and biomedical implants for organ replacement and repair. The wicking property and flexibility of threads also make them promising candidates for the creation of three-dimensional (3D) microfluidic circuits. In this paper, we report on thread-based microfluidic networks that interface intimately with biological tissues in three dimensions. We have also developed a suite of physical and chemical sensors integrated with microfluidic networks to monitor physiochemical tissue properties, all made from thread, for direct integration with tissues toward the realization of a thread-based diagnostic device (TDD) platform. The physical and chemical sensors are fabricated from nanomaterial-infused conductive threads and are connected to electronic circuitry using thread-based flexible interconnects for readout, signal conditioning, and wireless transmission. To demonstrate the suite of integrated sensors, we utilized TDD platforms to measure strain, as well as gastric and subcutaneous pH in vitro and in vivo.

Enhanced Intracellular siRNA Delivery using Bioreducible Lipid-Like Nanoparticles

A new library of lipid-like nanoparticles (lipidoids) comprising disulfide bond is developed for siRNA delivery. Bioreducible lipidoids deliver siRNA with greater efficiency than nonbioreducible lipidoids with similar chemical structures. A siRNA release investigation, as well as an intracellular siRNA trafficking study, reveals that the degradation of bioreducible lipidoid in a strongly reductive intracellular environment boosts siRNA release and enhances siRNA gene knockdown efficiency.

A Combinatorial Library of Unsaturated Lipidoids for Efficient Intracellular Gene Delivery

A combinatorial library of unsaturated lipidoids was synthesized through the Michael addition of amines to oleyl acrylamide. Their capability in facilitating in vitro gene delivery was evaluated by transfecting HeLa cells with EGFP-encoding plasmid DNA and mRNA. The preliminary screening results indicated that lipidoids with unsaturated oleyl tails are superior transfection agents compared to saturated lipidoids with n-octadecyl tails under the same conditions. The different transfection abilities of the unsaturated and saturated lipidioids were ascribed to the large, tightly packed lipoplexes of saturated lipidoids. The potential applications of the library of lipidoids were further expanded by looking at their ability to transfect fibroblasts as well as different cancerous cell lines.

DOPE facilitates quaternized lipidoids (QLDs) for in vitro DNA delivery

UNLABELLED This paper describes the synthesis of a combinatorial library of quaternized lipidoids (QLDs) and an evaluation of their abilities to facilitate in vitro DNA delivery. The QLDs alone showed low efficiency for DNA delivery. By formulating liposomes with a neutral helper lipid, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), the capability of QLDs for gene transfection is significantly enhanced due to the fusogenic properties of DOPE which facilitate endosomal escape and cargo delivery. We further optimized the liposome composition and DNA dose for gene transfection and investigated the structure-activity relationships of the lipidoid library in DNA delivery. FROM THE CLINICAL EDITOR This paper describes the synthesis and evaluation of a combinatorial library of quaternized lipidoids to facilitate in vitro DNA delivery, which occurs at a low level but can be enhanced with DOPE. The authors also further optimized the liposome composition and DNA dose for delivery and investigated the structure-activity relationships of the lipidoid library.

The behavior of neuronal cells on tendon-derived collagen sheets as potential substrates for nerve regeneration.

Peripheral nervous system injuries result in a decreased quality of life, and generally require surgical intervention for repair. Currently, the gold standard of nerve autografting, based on the use of host tissue such as sensory nerves is suboptimal as it results in donor-site loss of function and requires a secondary surgery. Nerve guidance conduits fabricated from natural polymers such as collagen are a common alternative to bridge nerve defects. In the present work, tendon sections derived through a process named bioskiving were studied for their potential for use as a substrate to fabricate nerve guidance conduits. We show that cells such as rat Schwann cells adhere, proliferate, and align along the fibrous tendon substrate which has been shown to result in a more mature phenotype. Additionally we demonstrate that chick dorsal root ganglia explants cultured on the tendon grow to similar lengths compared to dorsal root ganglia cultured on collagen gels, but also grow in a more oriented manner on the tendon sections. These results show that tendon sections produced through bioskiving can support directional nerve growth and may be of use as a substrate for the fabrication of nerve guidance conduits.

Carbonic anhydrase generates a pH gradient in Bombyx mori silk glands

Silk is a protein of interest to both biological and industrial sciences. The silkworm, Bombyx mori, forms this protein into strong threads starting from soluble silk proteins using a number of biochemical and physical cues to allow the transition from liquid to fibrous silk. A pH gradient has been measured along the gland, but the methodology employed was not able to precisely determine the pH at specific regions of interest in the silk gland. Furthermore, the physiological mechanisms responsible for the generation of this pH gradient are unknown. In this study, concentric ion selective microelectrodes were used to determine the luminal pH of B. mori silk glands. A gradient from pH 8.2 to 7.2 was measured in the posterior silk gland, with a pH 7 throughout the middle silk gland, and a gradient from pH 6.8 to 6.2 in the beginning of the anterior silk gland where silk processing into fibers occurs. The small diameter of the most anterior region of the anterior silk gland prevented microelectrode access in this region. Using a histochemical method, the presence of active carbonic anhydrase was identified in the funnel and anterior silk gland of fifth instar larvae. The observed pH gradient collapsed upon addition of the carbonic anhydrase inhibitor methazolamide, confirming an essential role for this enzyme in pH regulation in the B. mori silk gland. Plastic embedding of whole silk glands allowed clear visualization of the morphology, including the identification of four distinct epithelial cell types in the gland and allowed correlations between silk gland morphology and silk stages of assembly related to the pH gradient. B. mori silk glands have four different epithelial cell types, one of which produces carbonic anhydrase. Carbonic anhydrase is necessary for the mechanism that generates an intraluminal pH gradient, which likely regulates the assembly of silk proteins and then the formation of fibers from soluble silk proteins. These new insights into native silk formation may lead to a more efficient production of artificial or regenerated silkworm silk fibers.

Slicing, stacking and rolling: fabrication of nanostructured collagen constructs from tendon sections.

A novel method for fabricating both multilayer stacked 2D and 3D tubular constructs composed of sheets of aligned collagen fibers is described. These structures are created by decellularizing native tendon and sectioning the material into thin sheets using a cryo-microtome. This fabrication method preserves the collagens natural strength as well as the fiber structure which would aid in directing aligned cell growth.

Biocompatibility and degradation of tendon-derived scaffolds.

Decellularized extracellular matrix has often been used as a biomaterial for tissue engineering applications. Its function, once implanted can be crucial to determining whether a tissue engineered construct will be successful, both in terms of how the material breaks down, and how the body reacts to the material’s presence in the first place. Collagen is one of the primary components of extracellular matrix and has been used for a number of biomedical applications. Scaffolds comprised of highly aligned collagen fibrils can be fabricated directly from decellularized tendon using a slicing, stacking, and rolling technique, to create two- and three-dimensional constructs. Here, the degradation characteristics of the material are evaluated in vitro, showing that chemical crosslinking can reduce degradation while maintaining fiber structure. In vivo, non-crosslinked and crosslinked samples are implanted, and their biological response and degradation evaluated through histological sectioning, trichrome staining, and immunohistochemical staining for macrophages. Non-crosslinked samples are rapidly degraded and lose fiber morphology while crosslinked samples retain both macroscopic structure as well as fiber orientation. The cellular response of both materials is also investigated. The in vivo response demonstrates that the decellularized tendon material is biocompatible, biodegradable and can be crosslinked to maintain surface features for extended periods of time in vivo. This study provides material characteristics for the use of decellularized tendon as biomaterial for tissue engineering.

Nanostructured Tendon-Derived Scaffolds for Enhanced Bone Regeneration by Human Adipose-Derived Stem Cells

Decellularized matrix-based scaffolds can induce enhanced tissue regeneration due to their biochemical, biophysical, and mechanical similarity to native tissues. In this study, we report a nanostructured decellularized tendon scaffold with aligned, nanofibrous structures to enhance osteogenic differentiation and in vivo bone formation of human adipose-derived stem cells (hADSCs). Using a bioskiving method, we prepared decellularized tendon scaffolds from tissue slices of bovine Achilles and neck tendons with or without fixation, and investigated the effects on physical and mechanical properties of decellularized tendon scaffolds, based on the types and concentrations of cross-linking agents. In general, we found that decellularized tendon scaffolds without fixative treatments were more effective in inducing osteogenic differentiation and mineralization of hADSCs in vitro. When non-cross-linked decellularized tendon scaffolds were applied together with hydroxyapatite for hADSC transplantation in critical-sized bone defects, they promoted bone-specific collagen deposition and mineralized bone formation 4 and 8 weeks after hADSC transplantation, compared to conventional collagen type I scaffolds. Interestingly, stacking of decellularized tendon scaffolds cultured with osteogenically committed hADSCs and those containing human cord blood-derived endothelial progenitor cells (hEPCs) induced vascularized bone regeneration in the defects 8 weeks after transplantation. Our study suggests that biomimetic nanostructured scaffolds made of decellularized tissue matrices can serve as functional tissue-engineering scaffolds for enhanced osteogenesis of stem cells.