Search

Measuring changes in enzymatic activity over time from small numbers of cells remains a significant technical challenge. In this work, a method for sampling the cytoplasm of cells is introduced to extract enzymes and measure their activity at multiple time points. A microfluidic device, termed the live cell analysis device (LCAD), is designed, where cells are cultured in microwell arrays fabricated on polymer membranes containing nanochannels. Localized electroporation of the cells opens transient pores in the cell membrane at the interface with the nanochannels, enabling extraction of enzymes into nanoliter-volume chambers. In the extraction chambers, the enzymes modify immobilized substrates, and their activity is quantified by self-assembled monolayers for matrix-assisted laser desorption/ionization (SAMDI) mass spectrometry. By employing the LCAD-SAMDI platform, protein delivery into cells is demonstrated. Next, it is shown that enzymes can be extracted, and their activity measured without a loss in viability. Lastly, cells are sampled at multiple time points to study changes in phosphatase activity in response to oxidation by hydrogen peroxide. With this unique sampling device and label-free assay format, the LCAD with SAMDI enables a powerful new method for monitoring the dynamics of cellular activity from small populations of cells.

Only a tiny fraction of the nanomedicine-design space has been explored, owing to the structural complexity of nanomedicines and the lack of relevant high-throughput synthesis and analysis methods. Here, we report a methodology for determining structure–activity relationships and design rules for spherical nucleic acids (SNAs) functioning as cancer-vaccine candidates. First, we identified ~1,000 candidate SNAs on the basis of reasonable ranges for 11 design parameters that can be systematically and independently varied to optimize SNA performance. Second, we developed a high-throughput method for making SNAs at the picomolar scale in a 384-well format, and used a mass spectrometry assay to rapidly measure SNA immune activation. Third, we used machine learning to quantitatively model SNA immune activation and identify the minimum number of SNAs needed to capture optimum structure–activity relationships for a given SNA library. Our methodology is general, can reduce the number of nanoparticles that need to be tested by an order of magnitude, and could serve as a screening tool for the development of nanoparticle therapeutics.

Biomaterials hold great promise in helping the adult brain regenerate and rebuild after trauma. Peptide amphiphiles (PA) are highly versatile biomaterials, gelling and forming macromolecular structures when exposed to physiological levels of electrolytes. We are here reporting on the first ever in vivo use of self-assembling peptide amphiphile carrying a Tenascin-C signal (E(2)Ten-C PA) for the re-direction of endogenous neuroblasts in the rodent brain. The PA forms highly aligned nanofibers, displaying the migratory sequence of Tenascin-C glycoprotein as epitope. In this in vivo work, we have formed in situ a gel of aligned peptide amphiphile nanofibers presenting a migratory Tenascin-C signal sequence in the ventral horn of the rostral migratory stream, creating a track reaching the neocortex. Seven days post-transplant, doublecortin positive cells were observed migrating inside and alongside the injected biomaterial, reaching the cortex. We observed a 24-fold increase in number of redirected neuroblasts for the E(2)Ten-C PA injected animals compared to control. We also found injecting the E(2)Ten-C PA to cause minimal neuroinflammatory response. Analyzing GFAP(+) astrocytes and Iba1(+) microglia activation, the PA does not elicit a stronger neuroinflammatory response than would be expected from a small needle stab wound. Re-directing endogenous neuroblasts and increasing the number of cells reaching a site of injury using peptide amphiphiles may open up new avenues for utilizing the pool of neuroblasts and neural stem cells within the adult brain for regenerating damaged brain tissue and replacing neurons lost to injury.

Populations of cells exhibit variations in biochemical activity, resulting from many factors including random stochastic variability in protein production, metabolic and cell-cycle states, regulatory mechanisms, and external signaling. The development of methods for the analysis of single cells has allowed for the measurement and understanding of this inherent heterogeneity, yet methods for measuring protein activities on the single-cell scale lag behind their genetic analysis counterparts and typically report on expression rather than activity. This paper presents an approach to measure protein tyrosine phosphatase (PTP) activity in individual cells using self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS). Using flow cytometry, individual cells are first sorted into a well plate containing lysis buffer and a phosphopeptide substrate. After lysis and incubation—during which the PTP enzymes act on the peptide substrate—the reaction substrate and product are immobilized onto arrays of self-assembled monolayers, which are then analyzed using mass spectrometry. PTP activities from thousands of individual cells were measured and their distributions analyzed. This work demonstrates a general method for measuring enzyme activities in lysates derived from individual cells and will contribute to the understanding of cellular heterogeneity in a variety of contexts.

Cell-based, high-throughput screening (HTS) assays are increasingly important tools used in drug discovery, but frequently rely on readouts of gene expression or phenotypic changes and require development of specialized, labeled reporters. Here a cell-based, label-free assay compatible with HTS is introduced that can report quantitatively on enzyme activities by measuring mass changes of substrates with matrix-assisted laser desorption/ionization mass spectrometry. The assay uses self-assembled monolayers to culture cells on arrays presenting substrates, which serve as reporters for a desired enzyme activity. Each spot of cells is treated with a compound, cultured and lysed, enabling endogenous enzymes to act on the immobilized peptide substrate. It is demonstrated that the assay can measure protein tyrosine phosphatase (PTP) activity from as few as five cells and a screen is described that identifies a compound that reduces PTP activity in cell lysates. This approach offers a valuable addition to the methods available for cell-based screening.

Regeneration of neural tissues will require regrowth of axons lost due to trauma or degeneration to reestablish neuronal connectivity. One approach toward this goal is to provide directional cues to neurons that can promote and guide neurite growth. Our laboratory previously reported the formation of aligned monodomain gels of peptide amphiphile (PA) nanofibers over macroscopic length scales. In this work, we modified these aligned scaffolds specifically to support neural cell growth and function. This was achieved by displaying extracellular matrix (ECM) derived bioactive peptide epitopes on the surface of aligned nanofibers of the monodomain gel. Presentation of IKVAV or RGDS epitopes enhanced the growth of neurites from neurons encapsulated in the scaffold, while the alignment guided these neurites along the direction of the nanofibers. After two weeks of culture in the scaffold, neurons displayed spontaneous electrical activity and established synaptic connections. Scaffolds encapsulating neural progenitor cells were formed in situ within the spinal cord and resulted in the growth of oriented processes in vivo. Moreover, dorsal root ganglion (DRG) cells demonstrated extensive migration inside the scaffold, with the direction of their movement guided by fiber orientation. The bioactive and macroscopically aligned scaffold investigated here and similar variants can potentially be tailored for use in neural tissue regeneration.

Biomimetic materials that display natural bioactive signals derived from extracellular matrix molecules like laminin and fibronectin hold promise for promoting regeneration of the nervous system. In this work, we investigated a biomimetic peptide amphiphile (PA) presenting a peptide derived from the extracellular glycoprotein tenascin-C, known to promote neurite outgrowth through interaction with β1 integrin. The tenascin-C mimetic PA (TN-C PA) was found to self-assemble into supramolecular nanofibers and was incorporated through co-assembly into PA gels formed by highly aligned nanofibers. TN-C PA content in these gels increased the length and number of neurites produced from neurons differentiated from encapsulated P19 cells. Furthermore, gels containing TN-C PA were found to increase migration of cells out of neurospheres cultured on gel coatings. These bioactive gels could serve as artificial matrix therapies in regions of neuronal loss to guide neural stem cells and promote through biochemical cues neurite extension after differentiation. One example of an important target would be their use as biomaterial therapies in spinal cord injury. Statement of Significance Tenascin-C is an important extracellular matrix molecule in the nervous system and has been shown to play a role in regenerating the spinal cord after injury and guiding neural progenitor cells during brain development, however, minimal research has been reported exploring the use of biomimetic biomaterials of tenascin-C. In this work, we describe a selfassembling biomaterial system in which peptide amphiphiles present a peptide derived from tenascin-C that promotes neurite outgrowth. Encapsulation of neurons in hydrogels of aligned nanofibers formed by tenascin-C-mimetic peptide amphiphiles resulted in enhanced neurite outgrowth. Additionally, these peptide amphiphiles promoted migration of neural progenitor cells cultured on nanofiber coatings. Tenascin-C biomimetic biomaterials such as the one described here have significant potential in neuroregenerative medicine.

Peripheral nerve injuries can result in lifelong disability. Primary coaptation is the treatment of choice when the gap between transected nerve ends is short. Long nerve gaps seen in more complex injuries often require autologous nerve grafts or nerve conduits implemented into the repair. Nerve grafts, however, cause morbidity and functional loss at donor sites, which are limited in number. Nerve conduits, in turn, lack an internal scaffold to support and guide axonal regeneration, resulting in decreased efficacy over longer nerve gap lengths. By comparison, peptide amphiphiles (PAs) are molecules that can self-assemble into nanofibers, which can be aligned to mimic the native architecture of peripheral nerve. As such, they represent a potential substrate for use in a bioengineered nerve graft substitute. To examine this, we cultured Schwann cells with bioactive PAs (RGDS-PA, IKVAV-PA) to determine their ability to attach to and proliferate within the biomaterial. Next, we devised a PA construct for use in a peripheral nerve critical sized defect model. Rat sciatic nerve defects were created and reconstructed with autologous nerve, PLGA conduits filled with various forms of aligned PAs, or left unrepaired. Motor and sensory recovery were determined and compared among groups. Our results demonstrate that Schwann cells are able to adhere to and proliferate in aligned PA gels, with greater efficacy in bioactive PAs compared to the backbone-PA alone. In vivo testing revealed recovery of motor and sensory function in animals treated with conduit/PA constructs comparable to animals treated with autologous nerve grafts. Functional recovery in conduit/PA and autologous graft groups was significantly faster than in animals treated with empty PLGA conduits. Histological examinations also demonstrated increased axonal and Schwann cell regeneration within the reconstructed nerve gap in animals treated with conduit/PA constructs. These results indicate that PA nanofibers may represent a promising biomaterial for use in bioengineered peripheral nerve repair. © 2014 Elsevier Ltd.

Peptide nanostructures are an exciting class of supramolecular systems that can be designed for novel therapies with great potential in advanced medicine. This paper reviews progress on nanostructures based on peptide amphiphiles capable of forming one-dimensional assemblies that emulate in structure the nanofibers present in extracellular matrices. These systems are highly tunable using supramolecular chemistry, and can be designed to signal cells directly with bioactive peptides. Peptide amphiphile nanofibers can also be used to multiplex functions through co-assembly and designed to deliver proteins, nucleic acids, drugs, or cells. We illustrate here the functionality of these systems describing their use in regenerative medicine of bone, cartilage, the nervous system, the cardiovascular system, and other tissues. In addition, we highlight recent work on the use of peptide amphiphile assemblies to create hierarchical biomimetic structures with order beyond the nanoscale, and also discuss the future prospects of these supramolecular systems.

The power of independence: When exhibited on the surface of self-assembling peptide-amphiphile nanofibers, the hydrophobic laminin-derived IKVAV epitope induced nanofiber bundling through interdigitation with neighboring fibers and thus decreased the bioactivity of the resulting materials. The inclusion of charged amino acids in the peptide amphiphiles disrupted the tendency to bundle and led to significantly enhanced neurite outgrowth.