Publications by authors named "Gregory T Tietjen"

24 Publications

  • Page 1 of 1

Lysis of cold-storage-induced microvascular obstructions for ex vivo revitalization of marginal human kidneys.

Am J Transplant 2021 01 5;21(1):161-173. Epub 2020 Jul 5.

Department of Surgery, Yale School of Medicine, New Haven, Connecticut, USA.

Thousands of kidneys from higher-risk donors are discarded annually because of the increased likelihood of complications posttransplant. Given the severe organ shortage, there is a critical need to improve utilization of these organs. To this end, normothermic machine perfusion (NMP) has emerged as a platform for ex vivo assessment and potential repair of marginal organs. In a recent study of 8 transplant-declined human kidneys on NMP, we discovered microvascular obstructions that impaired microvascular blood flow. However, the nature and physiologic impact of these lesions were unknown. Here, in a study of 39 human kidneys, we have identified that prolonged cold storage of human kidneys induces accumulation of fibrinogen within tubular epithelium. Restoration of normoxic conditions-either ex vivo during NMP or in vivo following transplant-triggered intravascular release of fibrinogen correlating with red blood cell aggregation and microvascular plugging. Combined delivery of plasminogen and tissue plasminogen activator during NMP lysed the plugs leading to a significant reduction in markers of renal injury, improvement in indicators of renal function, and improved delivery of vascular-targeted nanoparticles. Our study suggests a new mechanism of cold storage injury in marginal organs and provides a simple treatment with immediate translational potential.
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http://dx.doi.org/10.1111/ajt.16148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7775334PMC
January 2021

Ex vivo isolated human vessel perfusion system for the design and assessment of nanomedicines targeted to the endothelium.

Bioeng Transl Med 2020 May 28;5(2):e10154. Epub 2020 Jan 28.

Department of Surgery Yale School of Medicine New Haven Connecticut.

Endothelial cells play a central role in the process of inflammation. Their biologic relevance, as well as their accessibility to IV injected therapeutics, make them a strong candidate for treatment with molecularly-targeted nanomedicines. Typically, the properties of targeted nanomedicines are first optimized in vitro in cell culture and then in vivo in rodent models. While cultured cells are readily available for study, results obtained from isolated cells can lack relevance to more complex in vivo environments. On the other hand, the quantitative assays needed to determine the impact of nanoparticle design on targeting efficacy are difficult to perform in animal models. Moreover, results from animal models often translate poorly to human systems. To address the need for an improved testing platform, we developed an isolated vessel perfusion system to enable dynamic and quantitative study of vascular-targeted nanomedicines in readily obtainable human vessels isolated from umbilical cords or placenta. We show that this platform technology enables the evaluation of parameters that are critical to targeting efficacy (including flow rate, selection of targeting molecule, and temperature). Furthermore, biologic replicates can be easily produced by evaluating multiple vessel segments from the same human donor in independent, modular chambers. The chambers can also be adapted to house vessels of a variety of sizes, allowing for the subsequent study of vessel segments in vivo following transplantation into immunodeficient mice. We believe this perfusion system can help to address long-standing issues in endothelial targeted nanomedicines and thereby enable more effective clinical translation.
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http://dx.doi.org/10.1002/btm2.10154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7237142PMC
May 2020

High-throughput quantitative microscopy-based half-life measurements of intravenously injected agents.

Proc Natl Acad Sci U S A 2020 02 3;117(7):3502-3508. Epub 2020 Feb 3.

Department of Biomedical Engineering, Yale University, New Haven, CT 06511;

Accurate analysis of blood concentration and circulation half-life is an important consideration for any intravenously administered agent in preclinical development or for therapeutic application. However, the currently available tools to measure these parameters are laborious, expensive, and inefficient for handling multiple samples from complex multivariable experiments. Here we describe a robust high-throughput quantitative microscopy-based method to measure the blood concentration and circulation half-life of any fluorescently labeled agent using only a small (2 µL) amount of blood volume, enabling additional end-point measurements to be assessed in the same subject. To validate this method, we demonstrate its use to measure the circulation half-life in mice of two types of fluorescently labeled polymeric nanoparticles of different sizes and surface chemistries and of a much smaller fluorescently labeled monoclonal antibody. Furthermore, we demonstrate the improved accuracy of this method compared to previously described methods.
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http://dx.doi.org/10.1073/pnas.1915450117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035491PMC
February 2020

Quantitating Endosomal Escape of a Library of Polymers for mRNA Delivery.

Nano Lett 2020 02 31;20(2):1117-1123. Epub 2020 Jan 31.

Department of Biomedical Engineering , Yale University , New Haven , Connecticut 06511 , United States.

Endosomal escape is a key step for intracellular drug delivery of nucleic acids, but reliable and sensitive methods for its quantitation remain an unmet need. In order to rationally optimize the mRNA transfection efficiency of a library of polymeric materials, we designed a deactivated Renilla luciferase-derived molecular probe whose activity can be restored only in the cytosol. This probe can be coencapsulated with mRNA in the same delivery vehicle, thereby accurately measuring its endosomal escape efficiency. We examined a library of poly(amine--ester) (PACE) polymers with different end groups using this probe and observed a strong correlation between endosomal escape and transfection efficiency ( = 0.9334). In addition, we found that mRNA encapsulation efficiency and endosomal escape, but not uptake, were determinant factors for transfection efficiency. The polymers with high endosomal escape/transfection efficiency also showed good transfection efficiency , and mRNA expression was primarily observed in spleens after intravenous delivery. Together, our study suggests that the luciferase probe can be used as an effective tool to quantitate endosomal escape, which is essential for rational optimization of intracellular drug delivery systems.
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http://dx.doi.org/10.1021/acs.nanolett.9b04426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7195212PMC
February 2020

Differential functional roles of fibroblasts and pericytes in the formation of tissue-engineered microvascular networks in vitro.

NPJ Regen Med 2020 6;5. Epub 2020 Jan 6.

1Department of Medicine, Section of Nephrology, Yale University School of Medicine, New Haven, CT 06520 USA.

Formation of a perfusable microvascular network (μVN) is critical for tissue engineering of solid organs. Stromal cells can support endothelial cell (EC) self-assembly into a μVN, but distinct stromal cell populations may play different roles in this process. Here we describe the differential effects that two widely used stromal cell populations, fibroblasts (FBs) and pericytes (PCs), have on μVN formation. We examined the effects of adding defined stromal cell populations on the self-assembly of ECs derived from human endothelial colony forming cells (ECFCs) into perfusable μVNs in fibrin gels cast within a microfluidic chamber. ECs alone failed to fully assemble a perfusable μVN. Human lung FBs stimulated the formation of EC-lined μVNs within microfluidic devices. RNA-seq analysis suggested that FBs produce high levels of hepatocyte growth factor (HGF). Addition of recombinant HGF improved while the c-MET inhibitor, Capmatinib (INCB28060), reduced μVN formation within devices. Human placental PCs could not substitute for FBs, but in the presence of FBs, PCs closely associated with ECs, formed a common basement membrane, extended microfilaments intercellularly, and reduced microvessel diameters. Different stromal cell types provide different functions in microvessel assembly by ECs. FBs support μVN formation by providing paracrine growth factors whereas PCs directly interact with ECs to modify microvascular morphology.
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http://dx.doi.org/10.1038/s41536-019-0086-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6944695PMC
January 2020

Progenitor-derived human endothelial cells evade alloimmunity by CRISPR/Cas9-mediated complete ablation of MHC expression.

JCI Insight 2019 10 17;4(20). Epub 2019 Oct 17.

Department of Immunobiology, Yale School of Medicine, New Haven, Connecticut, USA.

Tissue engineering may address organ shortages currently limiting clinical transplantation. Off-the-shelf engineered vascularized organs will likely use allogeneic endothelial cells (ECs) to construct microvessels required for graft perfusion. Vasculogenic ECs can be differentiated from committed progenitors (human endothelial colony-forming cells or HECFCs) without risk of mutation or teratoma formation associated with reprogrammed stem cells. Like other ECs, these cells can express both class I and class II major histocompatibility complex (MHC) molecules, bind donor-specific antibody (DSA), activate alloreactive T effector memory cells, and initiate rejection in the absence of donor leukocytes. CRISPR/Cas9-mediated dual ablation of β2-microglobulin and class II transactivator (CIITA) in HECFC-derived ECs eliminates both class I and II MHC expression while retaining EC functions and vasculogenic potential. Importantly, dually ablated ECs no longer bind human DSA or activate allogeneic CD4+ effector memory T cells and are resistant to killing by CD8+ alloreactive cytotoxic T lymphocytes in vitro and in vivo. Despite absent class I MHC molecules, these ECs do not activate or elicit cytotoxic activity from allogeneic natural killer cells. These data suggest that HECFC-derived ECs lacking MHC molecule expression can be utilized for engineering vascularized grafts that evade allorejection.
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http://dx.doi.org/10.1172/jci.insight.129739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824302PMC
October 2019

Tailoring Biomimetic Phosphorylcholine-Containing Block Copolymers as Membrane-Targeting Cellular Rescue Agents.

Biomacromolecules 2019 09 19;20(9):3385-3391. Epub 2019 Aug 19.

Department of Chemistry, Institute for Biophysical Dynamics, James Franck Institute , The University of Chicago , Chicago , Illinois 60637 , United States.

Some synthetic polymers can block cell death when applied following an injury that would otherwise kill the cell. This cellular rescue occurs through interactions of the polymers with cell membranes. However, general principles for designing synthetic polymers to ensure strong, but nondisruptive, cell membrane targeting are not fully elucidated. Here, we tailored biomimetic phosphorylcholine-containing block copolymers to interact with cell membranes and determined their efficacy in blocking neuronal death following oxygen-glucose deprivation. By adjusting the hydrophilicity and membrane affinity of poly(2-methacryloyloxyethyl phosphorylcholine) (polyMPC)-based triblock copolymers, the surface active regime in which the copolymers function effectively as membrane-targeting cellular rescue agents was determined. We identified nonintrusive interactions between the polymer and the cell membrane that alter the collective dynamics of the membrane by inducing rigidification without disrupting lipid packing or membrane thickness. In general, our results open new avenues for biological applications of polyMPC-based polymers and provide an approach to designing membrane-targeting agents to block cell death after injury.
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http://dx.doi.org/10.1021/acs.biomac.9b00621DOI Listing
September 2019

ZFYVE21 is a complement-induced Rab5 effector that activates non-canonical NF-κB via phosphoinosotide remodeling of endosomes.

Nat Commun 2019 05 21;10(1):2247. Epub 2019 May 21.

Division of Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT, 06520, USA.

Complement promotes vascular inflammation in transplant organ rejection and connective tissue diseases. Here we identify ZFYVE21 as a complement-induced Rab5 effector that induces non-canonical NF-κB in endothelial cells (EC). In response to membrane attack complexes (MAC), ZFYVE21 is post-translationally stabilized on MAC+Rab5+ endosomes in a Rab5- and PI(3)P-dependent manner. ZFYVE21 promotes SMURF2-mediated polyubiquitinylation and proteasome-dependent degradation of endosome-associated PTEN to induce vesicular enrichment of PI(3,4,5)P3 and sequential recruitment of activated Akt and NF-κB-inducing kinase (NIK). Pharmacologic alteration of cellular phosphoinositide content with miltefosine reduces ZFYVE21 induction, EC activation, and allograft vasculopathy in a humanized mouse model. ZFYVE21 induction distinctly occurs in response to MAC and is detected in human renal and synovial tissues. Our data identifies ZFYVE21 as a Rab5 effector, defines a Rab5-ZFYVE21-SMURF2-pAkt axis by which it mediates EC activation, and demonstrates a role for this pathway in complement-mediated conditions.
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http://dx.doi.org/10.1038/s41467-019-10041-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6529429PMC
May 2019

Poly(amine-co-ester) nanoparticles for effective Nogo-B knockdown in the liver.

J Control Release 2019 06 1;304:259-267. Epub 2019 May 1.

Department of Biomedical Engineering, Yale University, New Haven, CT 06511, United States of America; Department of Chemical & Environmental Engineering, Yale University, New Haven, CT 06511, United States of America; Department of Cellular & Molecular Physiology, Yale School of Medicine, New Haven, CT 06510, United States of America; Department of Dermatology, Yale School of Medicine, New Haven, CT 06510, United States of America. Electronic address:

Degradable poly(amine-co-ester) (PACE) terpolymers hold tremendous promise for siRNA delivery because these materials can be formulated into delivery vehicles with highly efficient siRNA encapsulation, providing effective knockdown with low toxicity. Here, we demonstrate that PACE nanoparticles (NPs) provide substantial protein knockdown in human embryonic kidney cells (HEK293) and hard-to-transfect primary human umbilical vein endothelial cells (HUVECs). After intravenous administration, NPs of solid PACE (sPACE)-synthesized with high monomer content of a hydrophobic lactone-accumulated in the liver and, to a lesser extent, in other tissues. Within the liver, a substantial fraction of sPACE NPs were phagocytosed by liver macrophages, while a smaller fraction of NPs accumulated in hepatic stellate cells and liver sinusoidal endothelial cells, suggesting that sPACE NPs could deliver siRNA to diverse cell populations within the liver. To test this hypothesis, we loaded sPACE NPs with siRNA designed to knockdown Nogo-B, a protein that has been implicated in the progression of alcoholic liver disease and liver fibrosis. These sPACE:siRNA NPs produced up to 60% Nogo-B protein suppression in the liver after systemic administration. We demonstrate that sPACE NPs can effectively deliver siRNA therapeutics to the liver to mediate protein knockdown in vivo.
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http://dx.doi.org/10.1016/j.jconrel.2019.04.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6613984PMC
June 2019

Sensitivity of peripheral membrane proteins to the membrane context: A case study of phosphatidylserine and the TIM proteins.

Biochim Biophys Acta Biomembr 2018 10 18;1860(10):2126-2133. Epub 2018 Jun 18.

Program in Biophysical Sciences, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, United States of America; Department of Chemistry, The University of Chicago, Chicago, IL, United States of America; James Franck Institute, The University of Chicago, Chicago, IL, United States of America. Electronic address:

There is a diverse class of peripheral membrane-binding proteins that specifically bind phosphatidylserine (PS), a lipid that signals apoptosis or cell fusion depending on the membrane context of its presentation. PS-receptors are specialized for particular PS-presenting pathways, indicating that they might be sensitive to the membrane context. In this review, we describe a combination of thermodynamic, structural, and computational techniques that can be used to investigate the mechanisms underlying this sensitivity. As an example, we focus on three PS-receptors of the T-cell Immunoglobulin and Mucin containing (TIM) protein family, which we have previously shown to differ in their sensitivity to PS surface density.
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http://dx.doi.org/10.1016/j.bbamem.2018.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6290684PMC
October 2018

Focus on Fundamentals: Achieving Effective Nanoparticle Targeting.

Trends Mol Med 2018 07 5;24(7):598-606. Epub 2018 Jun 5.

Department of Immunobiology, Yale School of Medicine, New Haven, CT 06510, USA.

Successful molecular targeting of nanoparticle drug carriers can enhance therapeutic specificity and reduce systemic toxicity. Typically, ligands specific for cognate receptors expressed on the intended target cell type are conjugated to the nanoparticle surface. This approach, often called active targeting, seems to imply that the conjugated ligand imbues the nanoparticle with homing capacity. However, ligand-receptor interactions are mediated by short-range forces and cannot produce magnetic-like attraction over larger distances. Successful targeting actually involves two key characteristics: contact of the nanoparticle with the intended target cell and subsequent ligand-mediated retention at the site. Here we propose a conceptual framework, based on recent literature combined with basic principles of molecular interactions, to guide rational design of nanoparticle targeting strategies.
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http://dx.doi.org/10.1016/j.molmed.2018.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028308PMC
July 2018

A "top-down" approach to actuate poly(amine-co-ester) terpolymers for potent and safe mRNA delivery.

Biomaterials 2018 09 25;176:122-130. Epub 2018 May 25.

Department of Biomedical Engineering, Yale University, New Haven, CT, 06511, USA; Department of Chemical and Environmental Engineering, Yale University, New Haven, CT, 06511, USA. Electronic address:

Gene delivery is known to be a complicated multi-step biological process. It has been observed that subtle differences in the structure and properties of polymeric materials used for gene delivery can lead to dramatic differences in transfection efficiency. Therefore, screening of properties is pivotal to optimizing the polymer. So far, most polymeric materials are built in a "bottom-up" manner, i.e. synthesized from monomers that allow modification of polymer composition or structural factors. With this method, we previously synthesized and screened a library of biodegradable poly(amine-co-ester) (PACE) terpolymers for optimized DNA delivery. However, it can be tedious and time consuming to synthesize a polymer library for screening, particularly when small changes of a factor need to be tested, when multiple factors are involved, and when the effects of different factors are synergistic. In the present work, we evaluate the potential of PACE to deliver mRNA. After observing that mRNA transfection efficiency was highly dependent on both end group composition and molecular weight (MW) of PACE in a synergistic manner, we developed a "top-down" process we called actuation, to simultaneously vary these two factors. Some of the actuated PACE (aPACE) materials presented superior mRNA delivery properties compared to regular PACE, with up to a 10-fold-increase in mRNA transfection efficiency in vitro. Moreover, when aPACE was used to deliver mRNA coding for erythropoietin (EPO) in vivo, it produced high levels of EPO in the blood for up to 48 h without inducing systemic toxicity. This polymer constitutes a new delivery vehicle for mRNA-based treatments that provides safe yet potent protein production.
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http://dx.doi.org/10.1016/j.biomaterials.2018.05.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6038928PMC
September 2018

The future of marginal kidney repair in the context of normothermic machine perfusion.

Am J Transplant 2018 10 2;18(10):2400-2408. Epub 2018 Jul 2.

Department of Surgery, University of Cambridge, Cambridge, UK.

Normothermic machine perfusion (NMP) is a technique that utilizes extracorporeal membrane oxygenation to recondition and repair kidneys at near body temperature prior to transplantation. The application of this new technology has been fueled by a significant increase in the use of the kidneys that were donated after cardiac death, which are more susceptible to ischemic injury. Preliminary results indicate that NMP itself may be able to repair marginal organs prior to transplantation. In addition, NMP serves as a platform for delivery of therapeutics. The isolated setting of NMP obviates problems of targeting a particular therapy to an intended organ and has the potential to reduce the harmful effects of systemic drug delivery. There are a number of emerging therapies that have shown promise in this platform. Nutrients, therapeutic gases, mesenchymal stromal cells, gene therapies, and nanoparticles, a newly explored modality, have been successfully delivered during NMP. These technologies may be effective at blocking multiple mechanisms of ischemia- reperfusion injury (IRI) and improving renal transplant outcomes. This review addresses the mechanisms of renal IRI, examines the potential for NMP as a platform for pretransplant allograft modulation, and discusses the introduction of various therapies in this setting.
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http://dx.doi.org/10.1111/ajt.14963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175453PMC
October 2018

Interferon-γ converts human microvascular pericytes into negative regulators of alloimmunity through induction of indoleamine 2,3-dioxygenase 1.

JCI Insight 2018 03 8;3(5). Epub 2018 Mar 8.

Department of Immunobiology, and.

Early acute rejection of human allografts is mediated by circulating alloreactive host effector memory T cells (TEM). TEM infiltration typically occurs across graft postcapillary venules and involves sequential interactions with graft-derived endothelial cells (ECs) and pericytes (PCs). While the role of ECs in allograft rejection has been extensively studied, contributions of PCs to this process are largely unknown. This study aimed to characterize the effects and mechanisms of interactions between human PCs and allogeneic TEM. We report that unstimulated PCs, like ECs, can directly present alloantigen to TEM, but while IFN-γ-activated ECs (γ-ECs) show increased ability to stimulate alloreactive T cells, IFN-γ-activated PCs (γ-PCs) instead suppress TEM proliferation but not cytokine production or signaling. RNA sequencing analysis of PCs, γ-PCs, ECs, and γ-ECs reveal induction of indoleamine 2,3-dioxygenase 1 (IDO1) in γ-PCs to significantly higher levels than in γ-ECs that correlates with tryptophan depletion in vitro. Consistently, shRNA knockdown of IDO1 markedly reduces γ-PC-mediated immunoregulatory effects. Furthermore, human PCs express IDO1 in a skin allograft rejection humanized mouse model and in human renal allografts with acute T cell-mediated rejection. We conclude that immunosuppressive properties of human PCs are not intrinsic but instead result from IFN-γ-induced IDO1-mediated tryptophan depletion.
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http://dx.doi.org/10.1172/jci.insight.97881DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5922286PMC
March 2018

Nanoparticle targeting to the endothelium during normothermic machine perfusion of human kidneys.

Sci Transl Med 2017 11;9(418)

Department of Immunobiology, Yale University, New Haven, CT 06520, USA.

Ex vivo normothermic machine perfusion (NMP) is a new clinical strategy to assess and resuscitate organs likely to be declined for transplantation, thereby increasing the number of viable organs available. Short periods of NMP provide a window of opportunity to deliver therapeutics directly to the organ and, in particular, to the vascular endothelial cells (ECs) that constitute the first point of contact with the recipient's immune system. ECs are the primary targets of both ischemia-reperfusion injury and damage from preformed antidonor antibodies, and reduction of perioperative EC injury could have long-term benefits by reducing the intensity of the host's alloimmune response. Using NMP to administer therapeutics directly to the graft avoids many of the limitations associated with systemic drug delivery. We have previously shown that polymeric nanoparticles (NPs) can serve as depots for long-term drug release, but ensuring robust NP accumulation within a target cell type (graft ECs in this case) remains a fundamental challenge of nanomedicine. We show that surface conjugation of an anti-CD31 antibody enhances targeting of NPs to graft ECs of human kidneys undergoing NMP. Using a two-color quantitative microscopy approach, we demonstrate that targeting can enhance EC accumulation by about 5- to 10-fold or higher in discrete regions of the renal vasculature. In addition, our studies reveal that NPs can also nonspecifically accumulate within obstructed regions of the vasculature that are poorly perfused. These quantitative preclinical human studies demonstrate the therapeutic potential for targeted nanomedicines delivered during ex vivo NMP.
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http://dx.doi.org/10.1126/scitranslmed.aam6764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931373PMC
November 2017

Coupling X-Ray Reflectivity and In Silico Binding to Yield Dynamics of Membrane Recognition by Tim1.

Biophys J 2017 Oct;113(7):1505-1519

Program in Biophysical Sciences, Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois; Department of Chemistry, The University of Chicago, Chicago, Illinois; James Franck Institute, The University of Chicago, Chicago, Illinois. Electronic address:

The dynamic nature of lipid membranes presents significant challenges with respect to understanding the molecular basis of protein/membrane interactions. Consequently, there is relatively little known about the structural mechanisms by which membrane-binding proteins might distinguish subtle variations in lipid membrane composition and/or structure. We have previously developed a multidisciplinary approach that combines molecular dynamics simulation with interfacial x-ray scattering experiments to produce an atomistic model for phosphatidylserine recognition by the immune receptor Tim4. However, this approach requires a previously determined protein crystal structure in a membrane-bound conformation. Tim1, a Tim4 homolog with distinct differences in both immunological function and sensitivity to membrane composition, was crystalized in a closed-loop conformation that is unlikely to support membrane binding. Here we have used a previously described highly mobile membrane mimetic membrane in combination with a conventional lipid bilayer model to generate a membrane-bound configuration of Tim1 in silico. This refined structure provided a significantly improved fit of experimental x-ray reflectivity data. Moreover, the coupling of the x-ray reflectivity analysis with both highly mobile membrane mimetic membranes and conventional lipid bilayer molecular dynamics simulations yielded a dynamic model of phosphatidylserine membrane recognition by Tim1 with atomic-level detail. In addition to providing, to our knowledge, new insights into the molecular mechanisms that distinguish the various Tim receptors, these results demonstrate that in silico membrane-binding simulations can remove the requirement that the existing crystal structure be in the membrane-bound conformation for effective x-ray reflectivity analysis. Consequently, this refined methodology has the potential for much broader applicability with respect to defining the atomistic details of membrane-binding proteins.
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http://dx.doi.org/10.1016/j.bpj.2017.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627149PMC
October 2017

Ex vivo pretreatment of human vessels with siRNA nanoparticles provides protein silencing in endothelial cells.

Nat Commun 2017 08 4;8(1):191. Epub 2017 Aug 4.

Department of Biomedical Engineering, Yale University, New Haven, CT, 06511, USA.

Human endothelial cells are initiators and targets of the rejection response. Pre-operative modification of endothelial cells by small interfering RNA transfection could shape the nature of the host response post-transplantation. Ablation of endothelial cell class II major histocompatibility complex molecules by small interfering RNA targeting of class II transactivator can reduce the capacity of human endothelial cells to recruit and activate alloreactive T cells. Here, we report the development of small interfering RNA-releasing poly(amine-co-ester) nanoparticles, distinguished by their high content of a hydrophobic lactone. We show that a single transfection of small interfering RNA targeting class II transactivator attenuates major histocompatibility complex class II expression on endothelial cells for at least 4 to 6 weeks after transplantation into immunodeficient mouse hosts. Furthermore, silencing of major histocompatibility complex class II reduces allogeneic T-cell responses in vitro and in vivo. These data suggest that poly(amine-co-ester) nanoparticles, potentially administered during ex vivo normothermic machine perfusion of human organs, could be used to modify endothelial cells with a sustained effect after transplantation.The use of gene silencing techniques in the treatment of post-transplantation host rejection is not long lasting and can have systemic effects. Here, the authors utilize a nanocarrier for siRNA for treatment of arteries ex vivo prior to implantation subsequently attenuating immune reaction in vivo.
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http://dx.doi.org/10.1038/s41467-017-00297-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5543113PMC
August 2017

Surface chemistry governs cellular tropism of nanoparticles in the brain.

Nat Commun 2017 05 19;8:15322. Epub 2017 May 19.

Department of Biomedical Engineering, Malone Engineering Center, Yale University, New Haven, Connecticut 06510, USA.

Nanoparticles are of long-standing interest for the treatment of neurological diseases such as glioblastoma. Most past work focused on methods to introduce nanoparticles into the brain, suggesting that reaching the brain interstitium will be sufficient to ensure therapeutic efficacy. However, optimized nanoparticle design for drug delivery to the central nervous system is limited by our understanding of their cellular deposition in the brain. Here, we investigated the cellular fate of poly(lactic acid) nanoparticles presenting different surface chemistries, after administration by convection-enhanced delivery. We demonstrate that nanoparticles with 'stealth' properties mostly avoid internalization by all cell types, but internalization can be enhanced by functionalization with bio-adhesive end-groups. We also show that association rates measured in cultured cells predict the extent of internalization of nanoparticles in cell populations. Finally, evaluating therapeutic efficacy in an orthotopic model of glioblastoma highlights the need to balance significant uptake without inducing adverse toxicity.
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http://dx.doi.org/10.1038/ncomms15322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5454541PMC
May 2017

Quantitative microscopy-based measurements of circulating nanoparticle concentration using microliter blood volumes.

Nanomedicine 2017 Aug 13;13(6):1863-1867. Epub 2017 Apr 13.

Department of Biomedical Engineering, Yale University, New Haven, CT, USA. Electronic address:

Nanoparticles (NPs) are potential drug delivery vehicles for treatment of a broad range of diseases. Intravenous (IV) administration, the most common form of delivery, is relatively non-invasive and provides (in theory) access throughout the circulatory system. However, in practice, many IV injected NPs are quickly eliminated by specialized phagocytes in the liver and spleen. Consequently, new materials have been developed with the capacity to significantly extend the circulating half-life of IV administered NPs. Unfortunately, current procedures for measuring circulation half-lives are often expensive, time consuming, and can require large blood volumes that are not compatible with mouse models of disease. Here we describe a simple and reliable procedure for measuring circulation half-life utilizing quantitative microscopy. This method requires only 2μL of blood and minimal sample preparation, yet provides robust quantitative results.
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http://dx.doi.org/10.1016/j.nano.2017.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725539PMC
August 2017

Enhancing potency of siRNA targeting fusion genes by optimization outside of target sequence.

Proc Natl Acad Sci U S A 2015 Dec 16;112(48):E6597-605. Epub 2015 Nov 16.

Department of Cellular & Molecular Physiology, Yale University, New Haven, CT 06511; Department of Biomedical Engineering, Yale University, New Haven, CT 06511

Canonical siRNA design algorithms have become remarkably effective at predicting favorable binding regions within a target mRNA, but in some cases (e.g., a fusion junction site) region choice is restricted. In these instances, alternative approaches are necessary to obtain a highly potent silencing molecule. Here we focus on strategies for rational optimization of two siRNAs that target the junction sites of fusion oncogenes BCR-ABL and TMPRSS2-ERG. We demonstrate that modifying the termini of these siRNAs with a terminal G-U wobble pair or a carefully selected pair of terminal asymmetry-enhancing mismatches can result in an increase in potency at low doses. Importantly, we observed that improvements in silencing at the mRNA level do not necessarily translate to reductions in protein level and/or cell death. Decline in protein level is also heavily influenced by targeted protein half-life, and delivery vehicle toxicity can confound measures of cell death due to silencing. Therefore, for BCR-ABL, which has a long protein half-life that is difficult to overcome using siRNA, we also developed a nontoxic transfection vector: poly(lactic-coglycolic acid) nanoparticles that release siRNA over many days. We show that this system can achieve effective killing of leukemic cells. These findings provide insights into the implications of siRNA sequence for potency and suggest strategies for the design of more effective therapeutic siRNA molecules. Furthermore, this work points to the importance of integrating studies of siRNA design and delivery, while heeding and addressing potential limitations such as restricted targetable mRNA regions, long protein half-lives, and nonspecific toxicities.
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http://dx.doi.org/10.1073/pnas.1517039112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4672813PMC
December 2015

Nanomedicine gets personal.

Sci Transl Med 2015 Nov;7(314):314fs47

Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA.

Companion nanoparticle imaging merges with drug delivery technologies toward personalized nanomedicine (Miller et al., this issue).
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http://dx.doi.org/10.1126/scitranslmed.aad6645DOI Listing
November 2015

A holistic approach to targeting disease with polymeric nanoparticles.

Nat Rev Drug Discov 2015 Apr 19;14(4):239-47. Epub 2015 Jan 19.

Department of Biomedical Engineering, Yale University, New Haven, Connecticut 06511, USA.

The primary goal of nanomedicine is to improve clinical outcomes. To this end, targeted nanoparticles are engineered to reduce non-productive distribution while improving diagnostic and therapeutic efficacy. Paradoxically, as this field has matured, the notion of targeting has been minimized to the concept of increasing the affinity of a nanoparticle for its target. This Opinion article outlines a holistic view of nanoparticle targeting, in which the route of administration, molecular characteristics and temporal control of the nanoparticles are potential design variables that must be considered simultaneously. This comprehensive vision for nanoparticle targeting will facilitate the integration of nanomedicines into clinical practice.
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http://dx.doi.org/10.1038/nrd4503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4451203PMC
April 2015

Molecular mechanism for differential recognition of membrane phosphatidylserine by the immune regulatory receptor Tim4.

Proc Natl Acad Sci U S A 2014 Apr 31;111(15):E1463-72. Epub 2014 Mar 31.

Program in Biophysical Sciences, Institute for Biophysical Dynamics, Department of Chemistry, and James Franck Institute, The University of Chicago, Chicago, IL 60637.

Recognition of phosphatidylserine (PS) lipids exposed on the extracellular leaflet of plasma membranes is implicated in both apoptotic cell removal and immune regulation. The PS receptor T cell immunoglobulin and mucin-domain-containing molecule 4 (Tim4) regulates T-cell immunity via phagocytosis of both apoptotic (high PS exposure) and nonapoptotic (intermediate PS exposure) activated T cells. The latter population must be removed at lower efficiency to sensitively control immune tolerance and memory cell population size, but the molecular basis for how Tim4 achieves this sensitivity is unknown. Using a combination of interfacial X-ray scattering, molecular dynamics simulations, and membrane binding assays, we demonstrate how Tim4 recognizes PS in the context of a lipid bilayer. Our data reveal that in addition to the known Ca(2+)-coordinated, single-PS binding pocket, Tim4 has four weaker sites of potential ionic interactions with PS lipids. This organization makes Tim4 sensitive to PS surface concentration in a manner capable of supporting differential recognition on the basis of PS exposure level. The structurally homologous, but functionally distinct, Tim1 and Tim3 are significantly less sensitive to PS surface density, likely reflecting the differences in immunological function between the Tim proteins. These results establish the potential for lipid membrane parameters, such as PS surface density, to play a critical role in facilitating selective recognition of PS-exposing cells. Furthermore, our multidisciplinary approach overcomes the difficulties associated with characterizing dynamic protein/membrane systems to reveal the molecular mechanisms underlying Tim4's recognition properties, and thereby provides an approach capable of providing atomic-level detail to uncover the nuances of protein/membrane interactions.
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http://dx.doi.org/10.1073/pnas.1320174111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3992656PMC
April 2014

An efficient method for the creation of tunable optical line traps via control of gradient and scattering forces.

Opt Express 2008 Jul;16(14):10341-8

Department of Physics and Materials Science Institute, University of Oregon, Eugene, OR97403-1274, USA.

Interparticle interaction energies and other useful physical characteristics can be extracted from the statistical properties of the motion of particles confined by an optical line trap. In practice, however, the potential energy landscape, U(x), imposed by the line provides an extra, and in general unknown, influence on particle dynamics. We describe a new class of line traps in which both the optical gradient and scattering forces acting on a trapped particle are designed to be linear functions of the line coordinate and in which their magnitude can be counterbalanced to yield a flat U(x). These traps are formed using approximate solutions to general relations concerning non-conservative optical forces that have been the subject of recent investigations [Y. Roichman, B. Sun, Y. Roichman, J. Amato-Grill, and D. G. Grier, Phys. Rev. Lett. 100, 013602-4 (2008).]. We implement the lines using holographic optical trapping and measure the forces acting on silica microspheres, demonstrating the tunability of the confining potential energy landscape. Furthermore, we show that our approach efficiently directs available laser power to the trap, in contrast to other methods.
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http://dx.doi.org/10.1364/oe.16.010341DOI Listing
July 2008