Synthesis and Activity of T-Cell Tumor-Directing MegaMolecules.

This paper describes the synthesis, characterization, and functional activity of 26 MegaMolecule-based bispecific antibody mimics for T-cell redirection toward HER2+ cancer cells. The work reports functional bispecific MegaMolecules that bind both receptor targets, and recruit and activate T-cells resulting in lysis of the target tumor cells. Changing the orientation of linkage between Fabs against either HER2 or CD3ε results in an approximately 150-fold range in potency. Increasing scaffold valency from Fab dimers up to tetramers improves the potency of the antibody mimics up to 5-fold, but with diminishing returns in effective dose beyond trimeric formats. Antibody mimics that present either one or two Fabs against either receptor target allows for initial engagement of one cell type over the other. Finally, the antibody mimics significantly reduce HER2+ tumor volumes in a humanized xenograft model of breast cancer.

HaloTag-Modified, Ferrocene Labeled Self-Assembled Monolayers for Protein Sensing.

Ferrocene (Fc)-based disulfide molecules of various lengths with amino acid scaffolds and alkane or oligo(phenylene-ethynylene) (OPE) bridges are used in a mixed SAM with a di-(ethylene oxide) terminal mercaptoundecanol diluent (PEG2). The relative height of the Fc redox reporter in the SAM is compared to determine if there are protective effects like antifouling and specific detection. The HaloTag-binding motif is used as a proof-of-concept to investigate the electrochemical response to the HaloTag protein due to its known covalent and fast linkage. When the Fc-SAMs are exposed to the HaloTag protein, there are an antifouling nature and more specific detection for the engulfed Fc-based molecules (C6tBu/Halo). The further out the Fc is from the SAM layer, the more nonspecific adsorption is detected. The double layer capacitance (CDL) has the smallest change for the C6tBu control (ΔCDL = -0.1 µF cm-2) showing antifouling properties and produces a large change (ΔCDL = 0.9 µF cm-2) as well as a shift in oxidation potential when the active C6Halo is exposed to the HaloTag protein (ΔE1/2 = 50 ± 10 mV). The remaining Fc molecules are partially in or outside the PEG2 layer, allowing more ion penetration/mobility even when the HaloTag protein is bound. Generally, a more disordered environment was observed for the Fc-based molecules when adding the HaloTag ligand, which is evident from a larger Efwhm and higher CDL. Desorption of the SAMs with sodium iodide (NaI) showed retention of the HaloTag protein bound with the corresponding ligand, whereas negative controls did not. Self-assembled monolayers for MALDI mass spectrometry (SAMDI-MS) were used as an orthogonal detection technique to show the qualitative binding of the HaloTag protein to the electrode. Together, these results provide insight into the antifouling and detection methods of engulfing the redox molecules in the SAM diluent.

Temporal Sampling of Enzymes from Live Cells by Localized Electroporation and Quantification of Activity by SAMDI Mass Spectrometry.

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.

Label-Free Assay of Protein Tyrosine Phosphatase Activity in Single Cells.

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. 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.

Traceless Immobilization of Analytes for High-Throughput Experiments with SAMDI Mass Spectrometry.

Label-free assays, and particularly those based on the combination of mass spectroscopy with surface chemistries, enable high-throughput experiments of a broad range of reactions. However, these methods can still require the incorporation of functional groups that allow immobilization of reactants and products to surfaces prior to analysis. In this paper, we report a traceless method for attaching molecules to a self-assembled monolayer for matrix-assisted laser desorption and ionization (SAMDI) mass spectrometry. This method uses monolayers that are functionalized with a 3-trifluoromethyl-3-phenyl-diazirine group that liberates nitrogen when irradiated and gives a carbene that inserts into a wide range of bonds to covalently immobilize molecules. Analysis of the monolayer with SAMDI then reveals peaks for each of the adducts formed from molecules in the sample. This method is applied to characterize a P450 drug metabolizing enzyme and to monitor a Suzuki-Miyaura coupling chemical reaction and is important because modification of the substrates with a functional group would alter their activities. This method will be important for high-throughput experiments in many areas, including reaction discovery and optimization.

Measuring Drug Metabolism Kinetics and Drug-Drug Interactions Using Self-Assembled Monolayers for Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry.

The competition of two drugs for the same metabolizing enzyme is a common mechanism for drug-drug interactions that can lead to altered kinetics in drug metabolism and altered elimination rates in vivo. With the prevalence of multidrug therapy, there is great potential for serious drug-drug interactions and adverse drug reactions. In an effort to prevent adverse drug reactions, the FDA mandates the evaluation of the potential for metabolic inhibition by every new chemical entity. Conventional methods for assaying drug metabolism (e.g., those based on HPLC) have been established for measuring drug-drug interactions; however, they are low-throughput. Here we describe an approach to measure the catalytic activity of CYP2C9 using the high-throughput technique self-assembled monolayers for matrix-assisted laser desorption-ionization (SAMDI) mass spectrometry. We measured the kinetics of CYP450 metabolism of the substrate, screened a set of drugs for inhibition of CYP2C9 and determined the Ki values for inhibitors. The throughput of this platform may enable drug metabolism and drug-drug interactions to be interrogated at a scale that cannot be achieved with current methods.

Cell-Based Screening: Cellular Assays with a Molecular Endpoint Measured by SAMDI Mass Spectrometry (Small 28/2016).

On page 3811, M. Mrksich and co-workers culture cells using self-assembled monolayers presenting cell adhesion ligands and enzyme substrates. A lysis buffer disrupts the cell membranes, releasing enzymes that modify the immobilized substrates. These modifications can be measured with SAMDI mass spectrometry, giving a high-throughput, cell-based assay.

Cellular Assays with a Molecular Endpoint Measured by SAMDI Mass Spectrometry.

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.

Supramolecular Nanofibers of Peptide Amphiphiles for Medicine.

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.

Surface potential modulation as a tool for mitigating challenges in SERS-based microneedle sensors.

Raman spectroscopic-based biosensing strategies are often complicated by low signal and the presence of multiple chemical species. While surface-enhanced Raman spectroscopy (SERS) nanostructured platforms are able to deliver high quality signals by focusing the electromagnetic field into a tight plasmonic hot-spot, it is not a generally applicable strategy as it often depends on the specific adsorption of the analyte of interest onto the SERS platform. This paper describes a strategy to address this challenge by using surface potential as a physical binding agent in the context of microneedle sensors. We show that the potential-dependent adsorption of different chemical species allows scrutinization of the contributions of different chemical species to the final spectrum, and that the ability to cyclically adsorb and desorb molecules from the surface enables efficient application of multivariate analysis methods. We demonstrate how the strategy can be used to mitigate potentially confounding phenomena, such as surface reactions, competitive adsorption and the presence of molecules with similar structures. In addition, this decomposition helps evaluate criteria to maximize the signal of one molecule with respect to others, offering new opportunities to enhance the measurement of analytes in the presence of interferants.

High-Throughput Microfluidics Platform for Intracellular Delivery and Sampling of Biomolecules from Live Cells.

Nondestructive cell membrane permeabilization systems enable the intracellular delivery of exogenous biomolecules for cell engineering tasks as well as the temporal sampling of cytosolic contents from live cells for the analysis of dynamic processes. Here, we report a microwell array format live-cell analysis device (LCAD) that can perform localized-electroporation induced membrane permeabilization, for cellular delivery or sampling, and directly interfaces with surface-based biosensors for analyzing the extracted contents. We demonstrate the capabilities of the LCAD via an automated high-throughput workflow for multimodal analysis of live-cell dynamics, consisting of quantitative measurements of enzyme activity using self-assembled monolayers for MALDI mass spectrometry (SAMDI) and deep-learning enhanced imaging and analysis. By combining a fabrication protocol that enables robust assembly and operation of multilayer devices with embedded gold electrodes and an automated imaging workflow, we successfully deliver functional molecules (plasmid and siRNA) into live cells at multiple time-points and track their effect on gene expression and cell morphology temporally. Furthermore, we report sampling performance enhancements, achieving saturation levels of protein tyrosine phosphatase activity measured from as few as 60 cells, and demonstrate control over the amount of sampled contents by optimization of electroporation parameters using a lumped model. Lastly, we investigate the implications of cell morphology on electroporation-induced sampling of fluorescent molecules using a deep-learning enhanced image analysis workflow.

Exploration of the nanomedicine-design space with high-throughput screening and machine learning.

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.

Combining SAMDI Mass Spectrometry and Peptide Arrays to Profile Phosphatase Activities.

Phosphatases, the enzymes responsible for dephosphorylating proteins, play critical roles in many cellular processes. While their importance is widely recognized, phosphatase activity and regulation remain poorly understood. Currently, there are few assays available that are capable of directly measuring phosphatase activity and specificity. We have previously introduced SAMDI (self-assembled monolayers on gold for matrix-assisted laser desorption/ionization) mass spectrometry as a technique to profile the substrate specificities of enzymes. SAMDI mass spectrometry assays are well suited to examine phosphatase activities and offer many advantages over current methods. This technique uses monolayers that terminate with a peptide or molecular enzyme substrate and allows for enzyme reactions to be performed on a surface that can easily be rinsed and analyzed by mass spectrometry without the need for analyte labeling. In this chapter, we describe the process of combining SAMDI mass spectrometry with peptide arrays to study the substrate specificities of two protein tyrosine phosphatases.

In vivo migration of endogenous brain progenitor cells guided by an injectable peptide amphiphile biomaterial.

Biomaterials hold great promise in helping the adult brain regenerate and rebuild after trauma. Peptide amphiphiles (PAs) 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 PA carrying a Tenascin-C signal (E2 Ten-C PA) for the redirection 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 PA 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 posttransplant, 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 E2 Ten-C PA-injected animals compared to control. We also found injecting the E2 Ten-C PA to cause minimal neuroinflammatory response. Analysing 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. Redirecting endogenous neuroblasts and increasing the number of cells reaching a site of injury using PAs 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.

Creating a stem cell niche in the inner ear using self-assembling peptide amphiphiles.

The use of human embryonic stem cells (hESCs) for regeneration of the spiral ganglion will require techniques for promoting otic neuronal progenitor (ONP) differentiation, anchoring of cells to anatomically appropriate and specific niches, and long-term cell survival after transplantation. In this study, we used self-assembling peptide amphiphile (PA) molecules that display an IKVAV epitope (IKVAV-PA) to create a niche for hESC-derived ONPs that supported neuronal differentiation and survival both in vitro and in vivo after transplantation into rodent inner ears. A feature of the IKVAV-PA gel is its ability to form organized nanofibers that promote directed neurite growth. Culture of hESC-derived ONPs in IKVAV-PA gels did not alter cell proliferation or viability. However, the presence of IKVAV-PA gels increased the number of cells expressing the neuronal marker beta-III tubulin and improved neurite extension. The self-assembly properties of the IKVAV-PA gel allowed it to be injected as a liquid into the inner ear to create a biophysical niche for transplanted cells after gelation in vivo. Injection of ONPs combined with IKVAV-PA into the modiolus of X-SCID rats increased survival and localization of the cells around the injection site compared to controls. Human cadaveric temporal bone studies demonstrated the technical feasibility of a transmastoid surgical approach for clinical intracochlear injection of the IKVAV-PA/ONP combination. Combining stem cell transplantation with injection of self-assembling PA gels to create a supportive niche may improve clinical approaches to spiral ganglion regeneration.

A tenascin-C mimetic peptide amphiphile nanofiber gel promotes neurite outgrowth and cell migration of neurosphere-derived cells.

UNLABELLED: 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 ß1 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.

Aligned neurite outgrowth and directed cell migration in self-assembled monodomain gels.

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.

ß1-Integrin and integrin linked kinase regulate astrocytic differentiation of neural stem cells.

Astrogliosis with glial scar formation after damage to the nervous system is a major impediment to axonal regeneration and functional recovery. The present study examined the role of ß1-integrin signaling in regulating astrocytic differentiation of neural stem cells. In the adult spinal cord ß1-integrin is expressed predominantly in the ependymal region where ependymal stem cells (ESCs) reside. ß1-integrin signaling suppressed astrocytic differentiation of both cultured ESCs and subventricular zone (SVZ) progenitor cells. Conditional knockout of ß1-integrin enhanced astrogliogenesis both by cultured ESCs and by SVZ progenitor cells. Previous studies have shown that injection into the injured spinal cord of a self-assembling peptide amphiphile that displays an IKVAV epitope (IKVAV-PA) limits glial scar formation and enhances functional recovery. Here we find that injection of IKVAV-PA induced high levels of ß1-integrin in ESCs in vivo, and that conditional knockout of ß1-integrin abolished the astroglial suppressive effects of IKVAV-PA in vitro. Injection into an injured spinal cord of PAs expressing two other epitopes known to interact with ß1-integrin, a Tenascin C epitope and the fibronectin epitope RGD, improved functional recovery comparable to the effects of IKVAV-PA. Finally we found that the effects of ß1-integrin signaling on astrogliosis are mediated by integrin linked kinase (ILK). These observations demonstrate an important role for ß1-integrin/ILK signaling in regulating astrogliosis from ESCs and suggest ILK as a potential target for limiting glial scar formation after nervous system injury.

A bioengineered peripheral nerve construct using aligned peptide amphiphile nanofibers.

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.