Self-Titrating Anticoagulant Nanocomplexes That Restore Homeostatic Regulation of the Coagulation Cascade

Antithrombotic therapy is a critical portion of the treatment regime for a number of life-threatening conditions, including cardiovascular disease, stroke, and cancer; yet, proper clinical management of anticoagulation remains a challenge because existing agents increase the propensity for bleeding in patients. Here, we describe the development of a bioresponsive peptide–polysaccharide nanocomplex that utilizes a negative feedback mechanism to self-titrate the release of anticoagulant in response to varying levels of coagulation activity. This nanoscale self-titrating activatable therapeutic, or nanoSTAT, consists of a cationic thrombin-cleavable peptide and heparin, an anionic polysaccharide and widely used clinical anticoagulant. Under nonthrombotic conditions, nanoSTATs circulate inactively, neither releasing anticoagulant nor significantly prolonging bleeding time. However, in response to life-threatening pulmonary embolism, nanoSTATs locally release their drug payload and prevent thrombosis. This autonomous negative feedback regulator may improve antithrombotic therapy by increasing the therapeutic window and decreasing the bleeding risk of anticoagulants.National Institutes of Health (U.S.) (R01CA124427-01)National Cancer Institute (U.S.) (U54CA119349)National Cancer Institute (U.S.) (U54CA119335)National Cancer Institute (U.S.) (Center of Cancer Nanotechnology Excellence at MIT-Harvard U54CA151884)David & Lucile Packard Foundation (Fellowship)David H. Koch Institute for Integrative Cancer Research at MIT (Marie D. and Pierre Casimir-Lambert Fund)National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051)MIT-Harvard Center of Cancer Nanotechnology Excellence (5 U54 CA151884-03)National Institutes of Health (U.S.). Medical Scientist Training Program (T32GM007753)National Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award F32CA159496-02)Burroughs Wellcome Fund (Career Award at the Scientific Interface

DNA-Encoded Antibody Libraries: A Unified Platform for Multiplexed Cell Sorting and Detection of Genes and Proteins

Whether for pathological examination or for fundamental biology studies, different classes of biomaterials and biomolecules are each measured from a different region of a typically heterogeneous tissue sample, thus introducing unavoidable sources of noise that are hard to quantitate. We describe the method of DNA-encoded antibody libraries (DEAL) for spatially multiplexed detection of ssDNAs and proteins as well as for cell sorting, all on the same diagnostic platform. DEAL is based upon the coupling of ssDNA oligomers onto antibodies which are then combined with the biological sample of interest. Spotted DNA arrays, which are found to inhibit biofouling, are utilized to spatially stratify the biomolecules or cells of interest. We demonstrate the DEAL technique for (1) the rapid detection of multiple proteins within a single microfluidic channel, and, with the additional step of electroless amplification of gold-nanoparticle labeled secondary antibodies, we establish a detection limit of 10 fM for the protein IL-2, 150 times more sensitive than the analogue ELISA; (2) the multiplexed, on-chip sorting of both immortalized cell lines and primary immune cells with an efficiency that exceeds surface-confined panning approaches; and (3) the co-detection of ssDNAs, proteins, and cell populations on the same platform

High-Density, Multiplexed Patterning of Cells at Single-Cell Resolution for Tissue Engineering and Other Applications

Surface chemistry meets tissue engineering: A novel surface-patterning approach for creating arrays of DNA squares is combined with a unique method for DNA-encoding of cells to construct dense arrays of distinct single cells. The cell patterns can be transferred from the substrate surface into thin hydrogel films, and these layers can be stacked to form 3D tissue constructs

Iterative in Situ Click Chemistry Assembles a Branched Capture Agent and Allosteric Inhibitor for Akt1

We describe the use of iterative in situ click chemistry to design an Akt-specific branched peptide triligand that is a drop-in replacement for monoclonal antibodies in multiple biochemical assays. Each peptide module in the branched structure makes unique contributions to affinity and/or specificity resulting in a 200 nM affinity ligand that efficiently immunoprecipitates Akt from cancer cell lysates and labels Akt in fixed cells. Our use of a small molecule to preinhibit Akt prior to screening resulted in low micromolar inhibitory potency and an allosteric mode of inhibition, which is evidenced through a series of competitive enzyme kinetic assays. To demonstrate the efficiency and selectivity of the protein-templated in situ click reaction, we developed a novel QPCR-based methodology that enabled a quantitative assessment of its yield. These results point to the potential for iterative in situ click chemistry to generate potent, synthetically accessible antibody replacements with novel inhibitory properties

Programmable probiotics for detection of cancer in urine

Rapid advances in the forward engineering of genetic circuitry in living cells has positioned synthetic biology as a potential means to solve numerous biomedical problems, including disease diagnosis and therapy. One challenge in exploiting synthetic biology for translational applications is to engineer microbes that are well tolerated by patients and seamlessly integrate with existing clinical methods. We use the safe and widely used probiotic Escherichia coli Nissle 1917 to develop an orally administered diagnostic that can noninvasively indicate the presence of liver metastasis by producing easily detectable signals in urine. Our microbial diagnostic generated a high-contrast urine signal through selective expansion in liver metastases (10[superscript 6]-fold enrichment) and high expression of a lacZ reporter maintained by engineering a stable plasmid system. The lacZ reporter cleaves a substrate to produce a small molecule that can be detected in urine. E. coli Nissle 1917 robustly colonized tumor tissue in rodent models of liver metastasis after oral delivery but did not colonize healthy organs or fibrotic liver tissue. We saw no deleterious health effects on the mice for more than 12 months after oral delivery. Our results demonstrate that probiotics can be programmed to safely and selectively deliver synthetic gene circuits to diseased tissue microenvironments in vivo.Ludwig Center for Molecular OncologyAmar G. Bose Research GrantSan Diego Center for Systems Biology (United States. National Institutes of Health Grant P50 GM085764)National Institute of General Medical Sciences (U.S.) (R01GM69811)National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051)National Institute of Environmental Health Sciences (Core Center Grant P30-ES002109)Misrock Foundation (Postdoctoral Fellowship)National Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award)Burroughs Wellcome Fund (Career Award at the Scientific Interface

Modular Nucleic Acid Assembled p/MHC Microarrays for Multiplexed Sorting of Antigen-Specific T Cells

The human immune system consists of a large number of T cells capable of recognizing and responding to antigens derived from various sources. The development of peptide-major histocompatibility (p/MHC) tetrameric complexes has enabled the direct detection of these antigen-specific T cells. With the goal of increasing throughput and multiplexing of T cell detection, protein microarrays spotted with defined p/MHC complexes have been reported, but studies have been limited due to the inherent instability and reproducibility of arrays produced via conventional spotted methods. Herein, we report on a platform for the detection of antigen-specific T cells on glass substrates that offers significant advantages over existing surface-bound schemes. In this approach, called “Nucleic Acid Cell Sorting (NACS)”, single-stranded DNA oligomers conjugated site-specifically to p/MHC tetramers are employed to immobilize p/MHC tetramers via hybridization to a complementary-printed substrate. Fully assembled p/MHC arrays are used to detect and enumerate T cells captured from cellular suspensions, including primary human T cells collected from cancer patients. NACS arrays outperform conventional spotted arrays assessed in key criteria such as repeatability and homogeneity. The versatility of employing DNA sequences for cell sorting is exploited to enable the programmed, selective release of target populations of immobilized T cells with restriction endonucleases for downstream analysis. Because of the performance, facile and modular assembly of p/MHC tetramer arrays, NACS holds promise as a versatile platform for multiplexed T cell detection

Integrated barcode chips for rapid, multiplexed analysis of proteins in microliter quantities of blood

As the tissue that contains the largest representation of the human proteome [1], blood is the most important fluid for clinical diagnostics [2, 3, 4]. However, although changes of plasma protein profiles reflect physiological or pathological conditions associated with many human diseases, only a handful of plasma proteins are routinely used in clinical tests. Reasons for this include the intrinsic complexity of the plasma proteome [1], the heterogeneity of human diseases and the rapid degradation of proteins in sampled blood [5]. We report an integrated microfluidic system, the integrated blood barcode chip that can sensitively sample a large panel of protein biomarkers over broad concentration ranges and within 10 min of sample collection. It enables on-chip blood separation and rapid measurement of a panel of plasma proteins from quantities of whole blood as small as those obtained by a finger prick. Our device holds potential for inexpensive, noninvasive and informative clinical diagnoses, particularly in point-of-care settings

Mass-encoded synthetic biomarkers for multiplexed urinary monitoring of disease

Biomarkers are increasingly important in the clinical management of complex diseases, yet our ability to discover new biomarkers remains limited by our dependence on endogenous molecules. Here we describe the development of exogenously administered `synthetic biomarkers' composed of mass-encoded peptides conjugated to nanoparticles that leverage intrinsic features of human disease and physiology for noninvasive urinary monitoring. These protease-sensitive agents perform three functions in vivo: target sites of disease, sample dysregulated protease activities and emit mass-encoded reporters into host urine for multiplexed detection by mass spectrometry. Using mouse models of liver fibrosis and cancer, we show that they can noninvasively monitor liver fibrosis and resolution without the need for invasive core biopsies and can substantially improve early detection of cancer compared with clinically used blood biomarkers. This approach of engineering synthetic biomarkers for multiplexed urinary monitoring should be broadly amenable to additional pathophysiological processes and to point-of-care diagnostics

A clinical microchip for evaluation of single immune cells reveals high functional heterogeneity in phenotypically similar T cells

Cellular immunity has an inherent high level of functional heterogeneity. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. We report a microfluidic platform designed for highly multiplexed (more than ten proteins), reliable, sample-efficient (~1 × 10^4 cells) and quantitative measurements of secreted proteins from single cells. We validated the platform by assessment of multiple inflammatory cytokines from lipopolysaccharide (LPS)-stimulated human macrophages and comparison to standard immunotechnologies. We applied the platform toward the ex vivo quantification of T cell polyfunctional diversity via the simultaneous measurement of a dozen effector molecules secreted from tumor antigen–specific cytotoxic T lymphocytes (CTLs) that were actively responding to tumor and compared against a cohort of healthy donor controls. We observed profound, yet focused, functional heterogeneity in active tumor antigen–specific CTLs, with the major functional phenotypes quantitatively identified. The platform represents a new and informative tool for immune monitoring and clinical assessment

Programmable probiotics for detection of cancer in urine

Rapid advances in the forward engineering of genetic circuitry in living cells has positioned synthetic biology as a potential means to solve numerous biomedical problems, including disease diagnosis and therapy. One challenge in exploiting synthetic biology for translational applications is to engineer microbes that are well tolerated by patients and seamlessly integrate with existing clinical methods. We use the safe and widely used probiotic Escherichia coli Nissle 1917 to develop an orally administered diagnostic that can noninvasively indicate the presence of liver metastasis by producing easily detectable signals in urine. Our microbial diagnostic generated a high-contrast urine signal through selective expansion in liver metastases (10 6 -fold enrichment) and high expression of a lacZ reporter maintained by engineering a stable plasmid system. The lacZ reporter cleaves a substrate to produce a small molecule that can be detected in urine. E. coli Nissle 1917 robustly colonized tumor tissue in rodent models of liver metastasis after oral delivery but did not colonize healthy organs or fibrotic liver tissue. We saw no deleterious health effects on the mice for more than 12 months after oral delivery. Our results demonstrate that probiotics can be programmed to safely and selectively deliver synthetic gene circuits to diseased tissue microenvironments in vivo