Publications by authors named "Paula T Hammond"

233 Publications

Temporal dynamics of intradermal cytokine response to tuberculin in Mycobacterium bovis BCG-vaccinated cattle using sampling microneedles.

Sci Rep 2021 Mar 29;11(1):7074. Epub 2021 Mar 29.

Department of Animal Sciences, Pennsylvania State University, University Park, PA, USA.

Bovine tuberculosis (bTB) is a disease of livestock with severe and worldwide economic, animal welfare and zoonotic consequences. Application of test-and-slaughter-based control polices reliant on tuberculin skin testing has been the mainstay of bTB control in cattle. However, little is known about the temporal development of the bovine tuberculin skin test response at the dermal sites of antigen injection. To fill this knowledge gap, we applied minimally-invasive sampling microneedles (SMNs) for intradermal sampling of interstitial fluid at the tuberculin skin test sites in Mycobacterium bovis BCG-vaccinated calves and determined the temporal dynamics of a panel of 15 cytokines and chemokines in situ and in the peripheral blood. The results reveal an orchestrated and coordinated cytokine and local chemokine response, identified IL-1RA as a potential soluble biomarker of a positive tuberculin skin response, and confirmed the utility of IFN-γ and IP-10 for bTB detection in blood-based assays. Together, the results highlight the utility of SMNs to identify novel biomarkers and provide mechanistic insights on the intradermal cytokine and chemokine responses associated with the tuberculin skin test in BCG-sensitized cattle.
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http://dx.doi.org/10.1038/s41598-021-86398-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007627PMC
March 2021

Oxidation-Responsive, Tunable Growth Factor Delivery from Polyelectrolyte-Coated Implants.

Adv Healthc Mater 2021 05 18;10(9):e2001941. Epub 2021 Mar 18.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

Polyelectrolyte multilayer (PEM) coatings, constructed on the surfaces of tissue engineering scaffolds using layer-by-layer assembly (LbL), promote sustained release of therapeutic molecules and have enabled regeneration of large-scale, pre-clinical bone defects. However, these systems primarily rely on non-specific hydrolysis of PEM components to foster drug release, and their pre-determined drug delivery schedules potentially limit future translation into innately heterogeneous patient populations. To trigger therapeutic delivery directly in response to local environmental stimuli, an LbL-compatible polycation solely degraded by cell-generated reactive oxygen species (ROS) was synthesized. These thioketal-based polymers were selectively cleaved by physiologic doses of ROS, stably incorporated into PEM films alongside growth factors, and facilitated tunable release of therapeutic bone morphogenetic protein-2 (BMP-2) upon oxidation. These coatings' sensitivity to oxidation was also dependent on the polyanions used in film construction, providing a simple method for enhancing ROS-mediated protein delivery in vitro. Correspondingly, when implanted in critically-sized rat calvarial defects, the most sensitive ROS-responsive coatings generated a 50% increase in bone regeneration compared with less sensitive formulations and demonstrated a nearly threefold extension in BMP-2 delivery half-life over conventional hydrolytically-sensitive coatings. These combined results highlight the potential of environmentally-responsive PEM coatings as tunable drug delivery systems for regenerative medicine.
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http://dx.doi.org/10.1002/adhm.202001941DOI Listing
May 2021

STING Activation with the cGAMP-STINGΔTM Signaling Complex.

Bio Protoc 2021 Feb 5;11(3):e3905. Epub 2021 Feb 5.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.

Activating the STING (stimulator of interferon genes) signaling pathway via administration of STING agonist cyclic GMP-AMP (cGAMP) has shown great promise in cancer immunotherapy. While state-of-the-art approaches have predominantly focused on the encapsulation of cGAMP into liposomes or polymersomes for cellular delivery, we discovered that the recombinant STING protein lacking the transmembrane domain (STINGΔTM) could be used as a functional carrier for cGAMP delivery and elicit type I IFN expression in STING-deficient cell lines. Using this approach, we generated anti-tumoral immunity in mouse melanoma and colon cancer models, providing a potential translatable platform for STING agonist-based immunotherapy. Here, we report the detailed STING activation protocols with cGAMP-STINGΔTM complex to assist researchers in further development of this approach. This protocol can also be easily expanded to other applications related to STING activation, such as control of various types of infections.
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http://dx.doi.org/10.21769/BioProtoc.3905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7952947PMC
February 2021

Lipidome-based Targeting of STAT3-driven Breast Cancer Cells Using Poly-l-glutamic Acid-coated Layer-by-Layer Nanoparticles.

Mol Cancer Ther 2021 04 3;20(4):726-738. Epub 2021 Feb 3.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.

The oncogenic transcription factor STAT3 is aberrantly activated in 70% of breast cancers, including nearly all triple-negative breast cancers (TNBCs). Because STAT3 is difficult to target directly, we considered whether metabolic changes driven by activated STAT3 could provide a therapeutic opportunity. We found that STAT3 prominently modulated several lipid classes, with most profound effects on N-acyl taurine and arachidonic acid, both of which are involved in plasma membrane remodeling. To exploit these metabolic changes therapeutically, we screened a library of layer-by-layer (LbL) nanoparticles (NPs) differing in the surface layer that modulates interactivity with the cell membrane. We found that poly-l-glutamic acid (PLE)-coated NPs bind to STAT3-transformed breast cancer cells with 50% greater efficiency than to nontransformed cells, and the heightened PLE-NP binding to TNBC cells was attenuated by STAT3 inhibition. This effect was also observed in densely packed three-dimensional breast cancer organoids. As STAT3-transformed cells show greater resistance to cytotoxic agents, we evaluated whether enhanced targeted delivery via PLE-NPs would provide a therapeutic advantage. We found that cisplatin-loaded PLE-NPs induced apoptosis of STAT3-driven cells at lower doses compared with both unencapsulated cisplatin and cisplatin-loaded nontargeted NPs. In addition, because radiation is commonly used in breast cancer treatment, and may alter cellular lipid distribution, we analyzed its effect on PLE-NP-cell binding. Irradiation of cells enhanced the STAT3-targeting properties of PLE-NPs in a dose-dependent manner, suggesting potential synergies between these therapeutic modalities. These findings suggest that cellular lipid changes driven by activated STAT3 may be exploited therapeutically using unique LbL NPs.
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http://dx.doi.org/10.1158/1535-7163.MCT-20-0505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026705PMC
April 2021

Surface Plasmon-Enhanced Short-Wave Infrared Fluorescence for Detecting Sub-Millimeter-Sized Tumors.

Adv Mater 2021 Feb 14;33(7):e2006057. Epub 2021 Jan 14.

Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

Short-wave infrared (SWIR, 900-1700 nm) enables in vivo imaging with high spatiotemporal resolution and penetration depth due to the reduced tissue autofluorescence and decreased photon scattering at long wavelengths. Although small organic SWIR dye molecules have excellent biocompatibility, they have been rarely exploited as compared to their inorganic counterparts, mainly due to their low quantum yield. To increase their brightness, in this work, the SWIR dye molecules are placed in close proximity to gold nanorods (AuNRs) for surface plasmon-enhanced emission. The fluorescence enhancement is optimized by controlling the dye-to-AuNR number ratio and up to ≈45-fold enhancement factor is achieved. In addition, the results indicate that the highest dye-to-AuNR number ratio gives the highest emission intensity per weight and this is used for synthesizing SWIR imaging probes using layer-by-layer (LbL) technique with polymer coating protection. Then, the SWIR imaging probes are applied for in vivo imaging of ovarian cancer and the surface coating effect on intratumor distribution of the imaging probes is investigated in two orthotopic ovarian cancer models. Lastly, it is demonstrated that the plasmon-enhanced SWIR imaging probe has great potential for fluorescence imaging-guided surgery by showing its capability to detect sub-millimeter-sized tumors.
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http://dx.doi.org/10.1002/adma.202006057DOI Listing
February 2021

Designer broad-spectrum polyimidazolium antibiotics.

Proc Natl Acad Sci U S A 2020 12 23;117(49):31376-31385. Epub 2020 Nov 23.

School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637459;

For a myriad of different reasons most antimicrobial peptides (AMPs) have failed to reach clinical application. Different AMPs have different shortcomings including but not limited to toxicity issues, potency, limited spectrum of activity, or reduced activity in situ. We synthesized several cationic peptide mimics, main-chain cationic polyimidazoliums (PIMs), and discovered that, although select PIMs show little acute mammalian cell toxicity, they are potent broad-spectrum antibiotics with activity against even pan-antibiotic-resistant gram-positive and gram-negative bacteria, and mycobacteria. We selected PIM1, a particularly potent PIM, for mechanistic studies. Our experiments indicate PIM1 binds bacterial cell membranes by hydrophobic and electrostatic interactions, enters cells, and ultimately kills bacteria. Unlike cationic AMPs, such as colistin (CST), PIM1 does not permeabilize cell membranes. We show that a membrane electric potential is required for PIM1 activity. In laboratory evolution experiments with the gram-positive we obtained PIM1-resistant isolates most of which had menaquinone mutations, and we found that a site-directed menaquinone mutation also conferred PIM1 resistance. In similar experiments with the gram-negative pathogen PIM1-resistant mutants did not emerge. Although PIM1 was efficacious as a topical agent, intraperitoneal administration of PIM1 in mice showed some toxicity. We synthesized a PIM1 derivative, PIM1D, which is less hydrophobic than PIM1. PIM1D did not show evidence of toxicity but retained antibacterial activity and showed efficacy in murine sepsis infections. Our evidence indicates the PIMs have potential as candidates for development of new drugs for treatment of pan-resistant bacterial infections.
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http://dx.doi.org/10.1073/pnas.2011024117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7739875PMC
December 2020

Genetically Defined Syngeneic Mouse Models of Ovarian Cancer as Tools for the Discovery of Combination Immunotherapy.

Cancer Discov 2021 Feb 6;11(2):384-407. Epub 2020 Nov 6.

Whitehead Institute for Biomedical Research, Cambridge, Massachusetts.

Despite advances in immuno-oncology, the relationship between tumor genotypes and response to immunotherapy remains poorly understood, particularly in high-grade serous tubo-ovarian carcinomas (HGSC). We developed a series of mouse models that carry genotypes of human HGSCs and grow in syngeneic immunocompetent hosts to address this gap. We transformed murine-fallopian tube epithelial cells to phenocopy homologous recombination-deficient tumors through a combined loss of and overexpression of and , which was contrasted with an identical model carrying wild-type . For homologous recombination-proficient tumors, we constructed genotypes combining loss of and overexpression of , and driven by or or overexpression. These lines form tumors recapitulating human disease, including genotype-driven responses to treatment, and enabled us to identify follistatin as a driver of resistance to checkpoint inhibitors. These data provide proof of concept that our models can identify new immunotherapy targets in HGSC. SIGNIFICANCE: We engineered a panel of murine fallopian tube epithelial cells bearing mutations typical of HGSC and capable of forming tumors in syngeneic immunocompetent hosts. These models recapitulate tumor microenvironments and drug responses characteristic of human disease. In a -overexpressing model, immune-checkpoint resistance was driven by follistatin..
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http://dx.doi.org/10.1158/2159-8290.CD-20-0818DOI Listing
February 2021

A modular polymer microbead angiogenesis scaffold to characterize the effects of adhesion ligand density on angiogenic sprouting.

Biomaterials 2021 01 16;264:120231. Epub 2020 Jul 16.

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. Electronic address:

Three-dimensional micro-physiological in vitro representations of human tissues and organs are emerging as important models of human pathophysiology and stand to make a significant impact on the process of drug discovery and development. An enduring need is to create microvascular networks within such 3D models, particularly for tissues with high metabolic demand such as the liver, pancreas, and the central nervous system. Here we report a facile approach to drive angiogenesis in nascent 3D culture models by embedding degradable hydrogel microbeads coated with induced pluripotent stem cell-derived endothelial cells (MB-iPSC-ECs) in a dense epithelial tissue. Specifically, we describe an approach to optimize microbead scaffold cues, independent of the external environment, by evaluating the iPSC-EC to microbead adhesion properties and how they influence the propensity of cells to both coat microbeads uniformly and undergo sprouting angiogenesis. We encapsulated MB-iPSC-ECs in PEG hydrogels, systematically varied the relative concentration of integrin-targeting peptide motifs in the microbead and surrounding environment, and found that an optimal microbead scaffold ligand regime of 0.1-0.25 mM promotes iPSC-EC monolayer formation and subsequent invasion into the synthetic matrix. We used these results to predict the regime of adhesion ligand required to promote angiogenesis of MB-iPSC-ECs in a co-culture hepatocarcinoma (HEPG2) microtissue model. This modular degradable microbead platform has the potential to promote angiogenic sprouting, which may ultimately support vascularization of a variety of cell-dense tissues.
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http://dx.doi.org/10.1016/j.biomaterials.2020.120231DOI Listing
January 2021

Rational design of multistage drug delivery vehicles for pulmonary RNA interference therapy.

Int J Pharm 2020 Dec 26;591:119989. Epub 2020 Oct 26.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, United States; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States; Institute for Soldier Nanotechnologies, Massachusetts Institute of Technology, Cambridge, MA, 02139, United States. Electronic address:

Small interfering RNA (siRNA) therapy has significant potential for the treatment of myriad diseases, including cancer. While intravenous routes of delivery have been found to be effective for efficient targeting to the liver, achieving high accumulations selectively in other organs, including lung tissues, can be a challenge. We demonstrate the rational design and engineering of a layer-by-layer (LbL) nanoparticle-containing aerosol that is able to achieve efficient, multistage delivery of siRNA in vitro. For the purpose, LbL nanoparticles were, for the first time, encapsulated in composite porous micro scale particles using a supercritical CO2-assisted spray drying (SASD) apparatus using chitosan as an excipient. Such particles exhibited aerodynamic properties highly favorable for pulmonary administration, and effective silencing of mutant KRAS in lung cancer cells derived from tumors of a non-small cell lung cancer (NSCLC) autochthonous model. Furthermore, efficient alveolar accumulation following inhalation in healthy mice was also observed, corroborating in vitro aerodynamic results, and opening new perspectives for further studies of effective lung therapies These results show that multistage aerosols assembled by supercritical CO-assisted spray drying can enable efficient RNA interference therapy of pulmonary diseases including lung cancer.
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http://dx.doi.org/10.1016/j.ijpharm.2020.119989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7970503PMC
December 2020

MFSD7C switches mitochondrial ATP synthesis to thermogenesis in response to heme.

Nat Commun 2020 09 24;11(1):4837. Epub 2020 Sep 24.

Koch Institute for Integrative Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

ATP synthesis and thermogenesis are two critical outputs of mitochondrial respiration. How these outputs are regulated to balance the cellular requirement for energy and heat is largely unknown. Here we show that major facilitator superfamily domain containing 7C (MFSD7C) uncouples mitochondrial respiration to switch ATP synthesis to thermogenesis in response to heme. When heme levels are low, MSFD7C promotes ATP synthesis by interacting with components of the electron transport chain (ETC) complexes III, IV, and V, and destabilizing sarcoendoplasmic reticulum Ca-ATPase 2b (SERCA2b). Upon heme binding to the N-terminal domain, MFSD7C dissociates from ETC components and SERCA2b, resulting in SERCA2b stabilization and thermogenesis. The heme-regulated switch between ATP synthesis and thermogenesis enables cells to match outputs of mitochondrial respiration to their metabolic state and nutrient supply, and represents a cell intrinsic mechanism to regulate mitochondrial energy metabolism.
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http://dx.doi.org/10.1038/s41467-020-18607-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515921PMC
September 2020

Plasticity of ether lipids promotes ferroptosis susceptibility and evasion.

Nature 2020 09 16;585(7826):603-608. Epub 2020 Sep 16.

Broad Institute, Cambridge, MA, USA.

Ferroptosis-an iron-dependent, non-apoptotic cell death process-is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers. The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions. However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR-Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome-ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis.
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http://dx.doi.org/10.1038/s41586-020-2732-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8051864PMC
September 2020

Enhancing chemotherapy response through augmented synthetic lethality by co-targeting nucleotide excision repair and cell-cycle checkpoints.

Nat Commun 2020 08 17;11(1):4124. Epub 2020 Aug 17.

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

In response to DNA damage, a synthetic lethal relationship exists between the cell cycle checkpoint kinase MK2 and the tumor suppressor p53. Here, we describe the concept of augmented synthetic lethality (ASL): depletion of a third gene product enhances a pre-existing synthetic lethal combination. We show that loss of the DNA repair protein XPA markedly augments the synthetic lethality between MK2 and p53, enhancing anti-tumor responses alone and in combination with cisplatin chemotherapy. Delivery of siRNA-peptide nanoplexes co-targeting MK2 and XPA to pre-existing p53-deficient tumors in a highly aggressive, immunocompetent mouse model of lung adenocarcinoma improves long-term survival and cisplatin response beyond those of the synthetic lethal p53 mutant/MK2 combination alone. These findings establish a mechanism for co-targeting DNA damage-induced cell cycle checkpoints in combination with repair of cisplatin-DNA lesions in vivo using RNAi nanocarriers, and motivate further exploration of ASL as a generalized strategy to improve cancer treatment.
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http://dx.doi.org/10.1038/s41467-020-17958-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431578PMC
August 2020

Electrostatic Conjugation of Nanoparticle Surfaces with Functional Peptide Motifs.

Bioconjug Chem 2020 09 11;31(9):2211-2219. Epub 2020 Aug 11.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, Massachusetts 02139, United States.

We report the surface functionalization of anionic layer by layer nanoparticles (LbL NPs) with cationic tumor-penetrating peptides (TPPs) via electrostatic adsorption while retaining particle stability and charge characteristics. This strategy eliminates the need for structural modifications of the peptide and enables facile functionalization of surface chemistries difficult to modify or inaccessible via covalent conjugation strategies. We show that both carboxylated and sulfated LbL NPs are able to accommodate linear and cyclic TPPs and used fluorescence-based detection assays to quantify peptide loading per NP. We also demonstrate that TPP activity is retained upon adsorption, implying sufficient numbers of peptides take on the appropriate surface orientation, enabling efficient uptake of functionalized NPs in vitro, as characterized via flow cytometry and deconvolution microscopy. Overall, we believe that this strategy will serve as a broadly applicable approach to impart electrostatically assembled NPs with bioactive peptide motifs.
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http://dx.doi.org/10.1021/acs.bioconjchem.0c00384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7895459PMC
September 2020

Cancer Cell Coating Nanoparticles for Optimal Tumor-Specific Cytokine Delivery.

ACS Nano 2020 09 5;14(9):11238-11253. Epub 2020 Sep 5.

Department of Chemical Engineering, Massachusetts Institute of Technology, 183 Memorial Drive, Cambridge, Massachusetts 02142, United States.

Although cytokine therapy is an attractive strategy to build a more robust immune response in tumors, cytokines have faced clinical failures due to toxicity. In particular, interleukin-12 has shown great clinical promise but was limited in translation because of systemic toxicity. In this study, we demonstrate an enhanced ability to reduce toxicity without affecting the efficacy of IL-12 therapy. We engineer the material properties of a NP to meet the enhanced demands for optimal cytokine delivery by using the layer-by-layer (LbL) approach. Importantly, using LbL, we demonstrate cell-level trafficking of NPs to preferentially localize to the cell's outer surface and act as a drug depot, which is required for optimal payload activity on neighboring cytokine membrane receptors. LbL-NPs showed efficacy against a tumor challenge in both colorectal and ovarian tumors at doses that were not tolerated when administered carrier-free.
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http://dx.doi.org/10.1021/acsnano.0c03109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530125PMC
September 2020

Self-assembled cGAMP-STINGΔTM signaling complex as a bioinspired platform for cGAMP delivery.

Sci Adv 2020 Jun 12;6(24):eaba7589. Epub 2020 Jun 12.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

The stimulator of interferon (IFN) genes (STING) pathway constitutes a highly important part of immune responses against various cancers and infections. Consequently, administration of STING agonists such as cyclic GMP-AMP (cGAMP) has been identified as a promising approach to target these diseases. In cancer cells, STING signaling is frequently impaired by epigenetic silencing of STING; hence, conventional delivery of only its agonist cGAMP may be insufficient to trigger STING signaling. In this work, while expression of STING lacking the transmembrane (TM) domain is known to be unresponsive to STING agonists and is dominant negative when coexpressed with the full-length STING inside cells, we observed that the recombinant TM-deficient STING protein complexed with cGAMP could effectively trigger STING signaling when delivered in vitro and in vivo, including in STING-deficient cell lines. Thus, this bioinspired method using TM-deficient STING may present a universally applicable platform for cGAMP delivery.
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http://dx.doi.org/10.1126/sciadv.aba7589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7292616PMC
June 2020

Approaches to Modulate the Chronic Wound Environment Using Localized Nucleic Acid Delivery.

Adv Wound Care (New Rochelle) 2021 Sep 7;10(9):503-528. Epub 2020 Jul 7.

Institute for Soldier Nanotechnologies, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

Nonhealing wounds have been the subject of decades of basic and clinical research. Despite new knowledge about the biology of impaired wound healing, little progress has been made in treating chronic wounds, leaving patients with few therapeutic options. Diabetic ulcers are a particularly common form of nonhealing wound. Recently, investigation of therapeutic nucleic acids (TNAs), including plasmid DNA, small interfering RNA, microRNA mimics, anti-microRNA oligonucleotides, messenger RNA, and antisense oligonucleotides, has created a new treatment strategy for chronic wounds. TNAs can modulate the wound toward a prohealing environment by targeting gene pathways associated with inflammation, proteases, cell motility, angiogenesis, epithelialization, and oxidative stress. A variety of delivery systems have been investigated for TNAs, including dendrimers, lipid nanoparticles (NPs), polymeric micelles, polyplexes, metal NPs, and hydrogels. This review summarizes recent developments in TNA delivery for therapeutic targets associated with chronic wounds, with an emphasis on diabetic ulcers. Translational potential of TNAs remains a key challenge; we highlight some drug delivery approaches for TNAs that may hold promise. We also describe current commercial efforts to locally deliver nucleic acids to modulate the wound environment. Localized nucleic acid delivery holds promise for the treatment of nonhealing chronic wounds. Future efforts to improve targeting of these nucleic acid therapies in the wound with both spatial and temporal control through drug delivery systems will be crucial to successful clinical translation.
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http://dx.doi.org/10.1089/wound.2020.1167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8260896PMC
September 2021

Layer-by-Layer Biomaterials for Drug Delivery.

Annu Rev Biomed Eng 2020 06 21;22:1-24. Epub 2020 Feb 21.

School of Engineering and Center for Biomedical Engineering, Brown University, Providence, Rhode Island 02912, USA; email:

Controlled drug delivery formulations have revolutionized treatments for a range of health conditions. Over decades of innovation, layer-by-layer (LbL) self-assembly has emerged as one of the most versatile fabrication methods used to develop multifunctional controlled drug release coatings. The numerous advantages of LbL include its ability to incorporate and preserve biological activity of therapeutic agents; coat multiple substrates of all scales (e.g., nanoparticles to implants); and exhibit tuned, targeted, and/or responsive drug release behavior. The functional behavior of LbL films can be related to their physicochemical properties. In this review, we highlight recent advances in the development of LbL-engineered biomaterials for drug delivery, demonstrating their potential in the fields of cancer therapy, microbial infection prevention and treatment, and directing cellular responses. We discuss the various advantages of LbL biomaterial design for a given application as demonstrated through in vitro and in vivo studies.
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http://dx.doi.org/10.1146/annurev-bioeng-060418-052350DOI Listing
June 2020

Tuning Nanoparticle Interactions with Ovarian Cancer through Layer-by-Layer Modification of Surface Chemistry.

ACS Nano 2020 02 10;14(2):2224-2237. Epub 2020 Feb 10.

Koch Institute for Integrative Cancer Research , Massachusetts Institute of Technology , 500 Main Street , Cambridge , Massachusetts 02142 , United States.

Nanoparticle surface chemistry is a fundamental engineering parameter that governs tumor-targeting activity. Electrostatic assembly generates controlled polyelectrolyte complexes through the process of adsorption and charge overcompensation utilizing synthetic polyions and natural biomacromolecules; it can yield films with distinctive hydration, charge, and presentation of functional groups. Here, we used electrostatic layer-by-layer (LbL) assembly to screen 10 different surface chemistries for their ability to preferentially target human ovarian cancer . Our screen identified that poly-l-aspartate, poly-l-glutamate, and hyaluronate-coated LbL nanoparticles have striking specificity for ovarian cancer, while sulfated poly(β-cyclodextrin) nanoparticles target noncancerous stromal cells. We validated top candidates for tumor-homing ability with a murine model of metastatic disease and with patient-derived ovarian cancer spheroids. Nanoparticle surface chemistry also influenced subcellular trafficking, indicating strategies to target the cell membrane, caveolae, and perinuclear vesicles. Our results confirm LbL is a powerful tool to systematically engineer nanoparticles and achieve specific targeting.
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http://dx.doi.org/10.1021/acsnano.9b09213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062411PMC
February 2020

Engineering Helical Modular Polypeptide-Based Hydrogels as Synthetic Extracellular Matrices for Cell Culture.

Biomacromolecules 2020 02 2;21(2):566-580. Epub 2020 Jan 2.

Expanding the toolkit of modular and functional synthetic material systems for biomimetic extracellular matrices (ECMs) is needed for achieving more predictable and characterizable cell culture. In the present study, we engineered a synthetic hydrogel system incorporating poly(γ-propargyl-l-glutamate) (PPLG), an -carboxy anhydride polypeptide with a unique α-helical secondary structure. PPLG macromers were cross-linked into poly(ethylene glycol) (PEG) networks to form hybrid polypeptide-PEG hydrogels. We compared the properties of PPLG-PEG to systems where the PPLG macromers were replaced with 8-arm PEG or poly(γ-propargyl-d,l-glutamate) (PPDLG), which has a flexible random-coil conformation. We evaluated each hydrogel system as synthetic ECMs for two-dimensional (2D) endothelial cell culture. Cells on PPLG-PEG displayed superior attachment and spreading at comparable adhesion ligand incorporation concentrations, demonstrating the unique benefit of combining the more rigid and hydrophobic α-helical PPLG within the more flexible and hydrophilic PEG matrix. The modular PPLG macromer is a promising building block for developing other types of PPLG-based hydrogels with favorable and tunable properties.
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http://dx.doi.org/10.1021/acs.biomac.9b01297DOI Listing
February 2020

Binary Targeting of siRNA to Hematologic Cancer Cells using Layer-by-Layer Nanoparticles.

Adv Funct Mater 2019 May 27;29(20). Epub 2019 Mar 27.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

Using siRNA therapeutics to treat hematologic malignancies has been unsuccessful because blood cancer cells exhibit remarkable resistance to standard transfection methods. Herein we report the successful delivery of siRNA therapeutics with a dual-targeted, layer-by-layer nanoparticle (LbL-NP). The LbL-NP protects siRNA from nucleases in the bloodstream by embedding it within polyelectrolyte layers that coat a polymeric core. The outermost layer consists of hyaluronic acid (a CD44-ligand) covalently conjugated to CD20 antibodies. The CD20/CD44 dual-targeting outer layer provides precise binding to blood cancer cells, followed by receptor-mediated endocytosis of the LbL-NP. We use this siRNA delivery platform to silence B-cell lymphoma 2 (BCL-2), a pro-survival protein, and . The dual-targeting approach significantly enhanced internalization of BCL-2 siRNA in lymphoma and leukemia cells, which led to significant downregulation of BCL-2 expression. Systemic administration of the dual-targeted, siRNA-loaded nanoparticle induced apoptosis and hampered proliferation of blood cancer cells both in cell culture and in orthotopic non-Hodgkin's lymphoma animal models. These results provide the basis for approaches to targeting blood-borne cancers and other diseases, and suggest that LbL nanoassemblies are a promising approach for delivering therapeutic siRNA to hematopoetic cell types that are known to evade transfection by other means.
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http://dx.doi.org/10.1002/adfm.201900018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6910249PMC
May 2019

Temporal release of a three-component protein subunit vaccine from polymer multilayers.

J Control Release 2020 01 20;317:130-141. Epub 2019 Nov 20.

Interdisciplinary Research Group in Infectious Diseases, Singapore-MIT Alliance for Research and Technology, Singapore; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. Electronic address:

Sustained antigen and adjuvant availability have been shown to improve antiviral immune responses following vaccination. Transcutaneous delivery of vaccines using microneedles has also shown promise and may be particularly relevant for mosquito-borne viruses. We aim to combine these traits to create a three-component Protein Subunit vaccine on Microneedle Arrays (PSMNs) for transcutaneous delivery using layer-by-layer (LbL) assembly. Polymer multilayer thin films were generated to co-deliver a model combination of three chemically distinct vaccine components, a dengue virus Envelope protein Domain III (EDIII) subunit antigen and two adjuvants, a double-stranded RNA (Poly (inosinic:cytidylic acid) (PolyI:C)) and an amphiphilic hexapeptide, Pam3CSK4. Following application of PSMNs to the skin, implanted thin films facilitated sustained and temporal release of individual vaccine components from polymer multilayers. By modulating LbL composition and architecture, component release profiles in the skin could be independently tuned to allow release of adjuvants and antigen from days up to two weeks. Uptake of antigen and adjuvant from implanted vaccine films by antigen-presenting cells was demonstrated using in vivo mouse and ex vivo human skin models. Overall, we believe that such modular vaccine strategies offer design principles for enhancing the immunogenicity of protein subunit vaccines.
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http://dx.doi.org/10.1016/j.jconrel.2019.11.022DOI Listing
January 2020

Theranostic Layer-by-Layer Nanoparticles for Simultaneous Tumor Detection and Gene Silencing.

Angew Chem Int Ed Engl 2020 02 7;59(7):2776-2783. Epub 2020 Jan 7.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

Layer-by-layer nanoparticles (NPs) are modular drug delivery vehicles that incorporate multiple functional materials through sequential deposition of polyelectrolytes onto charged nanoparticle cores. Herein, we combined the multicomponent features and tumor targeting capabilities of layer-by-layer assembly with functional biosensing peptides to create a new class of nanotheranostics. These NPs encapsulate a high weight percentage of siRNA while also carrying a synthetic biosensing peptide on the surface that is cleaved into a urinary reporter upon exposure to specific proteases overexpressed in the tumor microenvironment. Importantly, this biosensor reports back on a molecular signature characteristic to metastatic tumors and associated with poor prognosis, MMP9 protease overexpression. This nanotheranostic mediates noninvasive urinary-based diagnostics in mouse models of three different cancers with simultaneous gene silencing in flank and metastatic mouse models of ovarian cancer.
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http://dx.doi.org/10.1002/anie.201911762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002217PMC
February 2020

Enantiomeric glycosylated cationic block co-beta-peptides eradicate Staphylococcus aureus biofilms and antibiotic-tolerant persisters.

Nat Commun 2019 10 21;10(1):4792. Epub 2019 Oct 21.

School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore, 637459, Singapore.

The treatment of bacterial infections is hindered by the presence of biofilms and metabolically inactive persisters. Here, we report the synthesis of an enantiomeric block co-beta-peptide, poly(amido-D-glucose)-block-poly(beta-L-lysine), with high yield and purity by one-shot one-pot anionic-ring opening (co)polymerization. The co-beta-peptide is bactericidal against methicillin-resistant Staphylococcus aureus (MRSA), including replicating, biofilm and persister bacterial cells, and also disperses biofilm biomass. It is active towards community-acquired and hospital-associated MRSA strains which are resistant to multiple drugs including vancomycin and daptomycin. Its antibacterial activity is superior to that of vancomycin in MRSA mouse and human ex vivo skin infection models, with no acute in vivo toxicity in repeated dosing in mice at above therapeutic levels. The copolymer displays bacteria-activated surfactant-like properties, resulting from contact with the bacterial envelope. Our results indicate that this class of non-toxic molecule, effective against different bacterial sub-populations, has promising potential for the treatment of S. aureus infections.
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http://dx.doi.org/10.1038/s41467-019-12702-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6803644PMC
October 2019

Enhancing humoral immunity via sustained-release implantable microneedle patch vaccination.

Proc Natl Acad Sci U S A 2019 08 29;116(33):16473-16478. Epub 2019 Jul 29.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139;

Sustained exposure of lymphoid tissues to vaccine antigens promotes humoral immunity, but traditional bolus immunizations lead to rapid antigen clearance. We describe a technology to tailor vaccine kinetics in a needle-free platform translatable to human immunization. Solid pyramidal microneedle (MN) arrays were fabricated with silk fibroin protein tips encapsulating a stabilized HIV envelope trimer immunogen and adjuvant, supported on a dissolving polymer base. Upon brief skin application, vaccine-loaded silk tips are implanted in the epidermis/upper dermis where they release vaccine over a time period determined by the crystallinity of the silk matrix. Following MN immunization in mice, Env trimer was released over 2 wk in the skin, correlating with increased germinal center (GC) B cell responses, a ∼1,300-fold increase in serum IgG titers and a 16-fold increase in bone marrow (BM) plasma cells compared with bolus immunization. Thus, implantable MNs provide a practical means to substantially enhance humoral immunity to subunit vaccines.
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http://dx.doi.org/10.1073/pnas.1902179116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697788PMC
August 2019

Layer-by-layer nanoparticles for novel delivery of cisplatin and PARP inhibitors for platinum-based drug resistance therapy in ovarian cancer.

Bioeng Transl Med 2019 May 14;4(2):e10131. Epub 2019 Jun 14.

The Koch Institute for Integrative Cancer Research Massachusetts Institute of Technology (MIT) Cambridge MA, 02142.

Advanced staged high-grade serous ovarian cancer (HGSOC) is the leading cause of gynecological cancer death in the developed world, with 5-year survival rates of only 25-30% due to late-stage diagnosis and the shortcomings of platinum-based therapies. A Phase I clinical trial of a combination of free cisplatin and poly(ADP-ribose) polymerase inhibitors (PARPis) showed therapeutic benefit for HGSOC. In this study, we address the challenge of resistance to platinum-based therapy by developing a targeted delivery approach. Novel electrostatic layer-by-layer (LbL) liposomal nanoparticles (NPs) with a terminal hyaluronic acid layer that facilitates CD44 receptor targeting are designed for selective targeting of HGSOC cells; the liposomes can be formulated to contain both cisplatin and the PARPi drug within the liposomal core and bilayer. The therapeutic effectiveness of LbL NP-encapsulated cisplatin and PARPi alone and in combination was compared with the corresponding free drugs in luciferase and CD44-expressing OVCAR8 orthotopic xenografts in female nude mice. The NPs exhibited prolonged blood circulation half-life, mechanistic staged drug release and targeted codelivery of the therapeutic agents to HGSOC cells. Moreover, compared to the free drugs, the NPs resulted in significantly reduced tumor metastasis, extended survival, and moderated systemic toxicity.
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http://dx.doi.org/10.1002/btm2.10131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6584097PMC
May 2019

Microneedle-based intradermal delivery of stabilized dengue virus.

Bioeng Transl Med 2019 May 25;4(2):e10127. Epub 2019 Feb 25.

Infectious Diseases IRG Singapore-MIT Alliance for Research and Technology Singapore.

Current live-attenuated dengue vaccines require strict cold chain storage. Methods to preserve dengue virus (DENV) viability, which enable vaccines to be transported and administered at ambient temperatures, will be decisive towards the implementation of affordable global vaccination schemes with broad immunization coverage in resource-limited areas. We have developed a microneedle (MN)-based vaccine platform for the stabilization and intradermal delivery of live DENV from minimally invasive skin patches. Dengue virus-stabilized microneedle arrays (VSMN) were fabricated using saccharide-based formulation of virus and could be stored dry at ambient temperature up to 3 weeks with maintained virus viability. Following intradermal vaccination, VSMN-delivered DENV was shown to elicit strong neutralizing antibody responses and protection from viral challenge, comparable to that of the conventional liquid vaccine administered subcutaneously. This work supports the potential for MN-based dengue vaccine technology and the progression towards cold chain-independence. Dengue virus can be stabilized using saccharide-based formulations and coated on microneedle array vaccine patches for storage in dry state with preserved viability at ambient temperature (VSMN; virus-stabilized microneedle arrays).
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http://dx.doi.org/10.1002/btm2.10127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6584444PMC
May 2019

Broad-Spectrum Proteome Editing with an Engineered Bacterial Ubiquitin Ligase Mimic.

ACS Cent Sci 2019 May 10;5(5):852-866. Epub 2019 May 10.

Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, 120 Olin Hall, Ithaca, New York 14853, United States.

Manipulation of the ubiquitin-proteasome pathway to achieve targeted silencing of cellular proteins has emerged as a reliable and customizable strategy for remodeling the mammalian proteome. One such approach involves engineering bifunctional proteins called ubiquibodies that are comprised of a synthetic binding protein fused to an E3 ubiquitin ligase, thus enabling post-translational ubiquitination and degradation of a target protein independent of its function. Here, we have designed a panel of new ubiquibodies based on E3 ubiquitin ligase mimics from bacterial pathogens that are capable of effectively interfacing with the mammalian proteasomal degradation machinery for selective removal of proteins of interest. One of these, the effector protein IpaH9.8 fused to a fibronectin type III () monobody that specifically recognizes green fluorescent protein (GFP), was observed to potently eliminate GFP and its spectral derivatives as well as 15 different FP-tagged mammalian proteins that varied in size (27-179 kDa) and subcellular localization (cytoplasm, nucleus, membrane-associated, and transmembrane). To demonstrate therapeutically relevant delivery of ubiquibodies, we leveraged a bioinspired molecular assembly method whereby synthetic mRNA encoding the GFP-specific ubiquibody was coassembled with poly A binding proteins and packaged into nanosized complexes using biocompatible, structurally defined polypolypeptides bearing cationic amine side groups. The resulting nanoplexes delivered ubiquibody mRNA in a manner that caused efficient target depletion in cultured mammalian cells stably expressing GFP as well as in transgenic mice expressing GFP ubiquitously. Overall, our results suggest that IpaH9.8-based ubiquibodies are a highly modular proteome editing technology with the potential for pharmacologically modulating disease-causing proteins.
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http://dx.doi.org/10.1021/acscentsci.9b00127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6535771PMC
May 2019

The Future of Layer-by-Layer Assembly: A Tribute to ACS Nano Associate Editor Helmuth Möhwald.

ACS Nano 2019 06 24;13(6):6151-6169. Epub 2019 May 24.

Fachbereich Physik, CHyN , Universität Hamburg , 22607 Hamburg , Germany.

Layer-by-layer (LbL) assembly is a widely used tool for engineering materials and coatings. In this Perspective, dedicated to the memory of ACS Nano associate editor Prof. Dr. Helmuth Möhwald, we discuss the developments and applications that are to come in LbL assembly, focusing on coatings, bulk materials, membranes, nanocomposites, and delivery vehicles.
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http://dx.doi.org/10.1021/acsnano.9b03326DOI Listing
June 2019

Solution Conditions Tune and Optimize Loading of Therapeutic Polyelectrolytes into Layer-by-Layer Functionalized Liposomes.

ACS Nano 2019 05 18;13(5):5623-5634. Epub 2019 Apr 18.

Koch Institute for Integrative Cancer Research , Massachusetts Institute of Technology , 500 Main Street , Cambridge , Massachusetts 02142 , United States.

Layer-by-layer (LbL) nanoparticles offer great potential to the field of drug delivery, where these nanocomposites have been studied for their ability to deliver chemotherapeutic agents, small molecule inhibitors, and nucleic acids. Most exciting is their ability to encapsulate multiple functional elements, which allow nanocarriers to deliver complex combination therapies with staged release. However, relative to planar LbL constructs, colloidal LbL systems have not undergone extensive systematic studies that outline critical synthetic solution conditions needed for robust and efficient assembly. The multistaged process of adsorbing a series of materials onto a nanoscopic template is inherently complex, and facilitating the self-assembly of these materials depends on identifying proper solution conditions for each synthetic step and adsorbed material. Here, we focus on addressing some of the fundamental questions that must be answered in order to obtain a reliable and robust synthesis of nucleic acid-containing LbL liposomes. This includes a study of solution conditions, such as pH, ionic strength, salt composition, and valency, and their impact on the preparation of LbL nanoparticles. Our results provide insight into the selection of solution conditions to control the degree of ionization and the electrostatic screening length to suit the adsorption of nucleic acids and synthetic polypeptides. The optimization of these parameters led to a roughly 8-fold improvement in nucleic acid loading in LbL liposomes, indicating the importance of optimizing solution conditions in the preparation of therapeutic LbL nanoparticles. These results highlight the benefits of defining principles for constructing highly effective nanoparticle systems.
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http://dx.doi.org/10.1021/acsnano.9b00792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980385PMC
May 2019

Deep-tissue optical imaging of near cellular-sized features.

Sci Rep 2019 03 7;9(1):3873. Epub 2019 Mar 7.

Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

Detection of biological features at the cellular level with sufficient sensitivity in complex tissue remains a major challenge. To appreciate this challenge, this would require finding tens to hundreds of cells (a 0.1 mm tumor has ~125 cells), out of ~37 trillion cells in the human body. Near-infrared optical imaging holds promise for high-resolution, deep-tissue imaging, but is limited by autofluorescence and scattering. To date, the maximum reported depth using second-window near-infrared (NIR-II: 1000-1700 nm) fluorophores is 3.2 cm through tissue. Here, we design an NIR-II imaging system, "Detection of Optically Luminescent Probes using Hyperspectral and diffuse Imaging in Near-infrared" (DOLPHIN), that resolves these challenges. DOLPHIN achieves the following: (i) resolution of probes through up to 8 cm of tissue phantom; (ii) identification of spectral and scattering signatures of tissues without a priori knowledge of background or autofluorescence; and (iii) 3D reconstruction of live whole animals. Notably, we demonstrate noninvasive real-time tracking of a 0.1 mm-sized fluorophore through the gastrointestinal tract of a living mouse, which is beyond the detection limit of current imaging modalities.
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http://dx.doi.org/10.1038/s41598-019-39502-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6405836PMC
March 2019
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