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Nanotechnology provides platforms to deliver medical agents to specific cells. However, the nanoparticle's surface becomes covered with serum proteins in the blood after administration despite engineering efforts to protect it with targeting or blocking molecules. Here, we developed a strategy to identify the main interactions between nanoparticle-adsorbed proteins and a cell by integrating mass spectrometry with pooled genome screens and Search Tool for the Retrieval of Interacting Genes analysis. We found that the low-density lipoprotein (LDL) receptor was responsible for approximately 75% of serum-coated gold nanoparticle uptake in U-87 MG cells. Apolipoprotein B and complement C8 proteins on the nanoparticle mediated uptake through the LDL receptor. In vivo, nanoparticle accumulation correlated with LDL receptor expression in the organs of mice. A detailed understanding of how adsorbed serum proteins bind to cell receptors will lay the groundwork for controlling the delivery of nanoparticles at the molecular level to diseased tissues for therapeutic and diagnostic applications.

Nanoparticles need to navigate a complex microenvironment to target cells in solid tumors after extravasation. Diffusion is currently the accepted primary mechanism for nanoparticle distribution in tumors. However, the extracellular matrix can limit nanoparticle diffusion. Here, we identified tumor-associated macrophages as another key player in transporting and redistributing nanoparticles in the tumor microenvironment. We found tumor-associated macrophages actively migrate toward nanoparticles extravasated from the vessels, engulfing and redistributing them in the tumor stroma. The macrophages can carry the nanoparticles 2-5 times deeper in the tumor than passive diffusion. The amount of nanoparticles transported by the tumor-associated macrophages is size-dependent. Understanding the nanoparticle behavior after extravasation will provide strategies to engineer them to navigate the microenvironment for improved intratumoral targeting and therapeutic effectiveness.

Diagnostic assays are commonly performed in multiple steps, where reagents are added at specific times and concentrations into a reaction chamber. The reagents require storage, preparation, and addition in the correct sequence and amount. These steps rely on trained technicians and instrumentation to perform each task. The reliance on such resources hinders the use of these diagnostic assays by lay users. We developed a tablet that can sequentially introduce prequantified lyophilized diagnostic reagents at specific time points for a multistep assay. We designed the tablet to have multiple layers using cellulose-grade polymers, such as microcrystalline cellulose and hydroxypropyl cellulose. Our formulation allows each layer to dissolve at a controlled rate to introduce reagents into the solution sequentially. The release rate is controlled by modulating the compression force or chemical formulation of the layer. Controlling the reagent release time is important because different assays have specific times when reagents need to be added. As proof of concept, we demonstrated two different assays with our tablet system. Our tablet detected nucleic acid target (

The successful delivery of nanoparticles to solid tumors depends on their ability to pass through blood vessels and into the tumor microenvironment. Here, we discovered a subset of tumor endothelial cells that facilitate nanoparticle transport into solid tumors. We named these cells nanoparticle transport endothelial cells (N-TECs). We show that only 21% of tumor endothelial cells located on a small number of vessels are involved in transporting nanoparticles into the tumor microenvironment. N-TECs have an increased expression of genes related to nanoparticle transport and vessel permeability compared to other tumor endothelial cells. The N-TECs act as gatekeepers that determine the entry point, distribution, cell accessibility, and number of nanoparticles that enter the tumor microenvironment.

The ability to rapidly diagnose, track, and disseminate information for SARS-CoV-2 is critical to minimize its spread. Here, we engineered a portable smartphone-based quantum barcode serological assay device for real-time surveillance of patients infected with SARS-CoV-2. Our device achieved a clinical sensitivity of 90% and specificity of 100% for SARS-CoV-2, as compared to 34% and 100%, respectively, for lateral flow assays in a head-to-head comparison. The lateral flow assay misdiagnosed ∼2 out of 3 SARS-CoV-2 positive patients. Our quantum dot barcode device has ∼3 times greater clinical sensitivity because it is ∼140 times more analytically sensitive than lateral flow assays. Our device can diagnose SARS-CoV-2 at different sampling dates and infectious severity. We developed a databasing app to provide instantaneous results to inform patients, physicians, and public health agencies. This assay and device enable real-time surveillance of SARS-CoV-2 seroprevalence and potential immunity.

The rapid and accurate detection of antimicrobial resistance is critical to limiting the spread of infections and delivering effective treatments. Here, we developed a rapid, sensitive, and simple colorimetric nanodiagnostic platform to identify disease-causing pathogens and their associated antibiotic resistance genes within 2 h. The platform can detect bacteria from different biological samples (i.e., blood, wound swabs) with or without culturing. We validated the multicomponent nucleic acid enzyme-gold nanoparticle (MNAzyme-GNP) platform by screening patients with central line associated bloodstream infections and achieved a clinical sensitivity and specificity of 86% and 100%, respectively. We detected antibiotic resistance in methicillin-resistant Staphylococcus aureus (MRSA) in patient swabs with 90% clinical sensitivity and 95% clinical specificity. Finally, we identified mecA resistance genes in uncultured nasal, groin, axilla, and wound swabs from patients with 90% clinical sensitivity and 95% clinical specificity. The simplicity and versatility for detecting bacteria and antibiotic resistance markers make our platform attractive for the broad screening of microbial pathogens.

Nanoparticle delivery to solid tumours over the past ten years has stagnated at a median of 0.7% of the injected dose. Varying nanoparticle designs and strategies have yielded only minor improvements. Here we discovered a dose threshold for improving nanoparticle tumour delivery: 1 trillion nanoparticles in mice. Doses above this threshold overwhelmed Kupffer cell uptake rates, nonlinearly decreased liver clearance, prolonged circulation and increased nanoparticle tumour delivery. This enabled up to 12% tumour delivery efficiency and delivery to 93% of cells in tumours, and also improved the therapeutic efficacy of Caelyx/Doxil. This threshold was robust across different nanoparticle types, tumour models and studies across ten years of the literature. Our results have implications for human translation and highlight a simple, but powerful, principle for designing nanoparticle cancer treatments. Efficient nanoparticle delivery into tumours has been a challenge in the field. It is now shown that the efficiency can be improved substantially when the dose breaches a specific threshold.

Nanovaccines need to be transported to lymph node follicles to induce humoral immunity and generate neutralizing antibodies. Here, we discovered that subcapsular sinus macrophages play a barrier role to prevent nanovaccines from accessing lymph node follicles. This is illustrated by measuring the humoral immune responses after removing or functionally altering these cells in the nanovaccine transport process. We achieved up to 60 times more antigen-specific antibody production after suppressing subcapsular sinus macrophages. The degree of the enhanced antibody production is dependent on the nanovaccine dose and size, formulation, and administration time. We further found that pharmacological agents that disrupt the macrophage uptake function can be considered as adjuvants in vaccine development. Immunizing mice using nanovaccines formulated with these agents can induce more than 30 times higher antigen-specific antibody production compared to nanovaccines alone. These findings suggest that altering transport barriers to enable more of the nanovaccine to be delivered to the lymph node follicles for neutralizing antibody production is an effective strategy to boost vaccination.

Point-of-care diagnostic assays often involve multistep reactions, requiring a wide range of precise temperatures. Although precise heating is critical to performing these assays, it is challenging to provide it in an electricity-free format away from established infrastructure. Chemical heaters are electricity-free and use exothermic reactions. However, they are unsuitable for point-of-care multistep reactions because they sacrifice portability, have a narrow range of achievable temperatures, and long ramp-up times. Here we developed a miniature heater by modulating the lithium–water reaction kinetics using bubbles in a channel. Our heaters are up to 8,000 times smaller than current devices and can provide precise (within 5 °C) and tunable heating from 37 °C to 65 °C (∆T(RT) = 12 °C to 40 °C) with ramp-up times of a minute. We demonstrate field portablity and stability and show their use in an electricity-free multistep workflow that needs a range of temperatures. Ultimately, we envision providing better access to cutting edge biochemical techniques, including diagnostics, by making portable and electricity-free heating available at any location.

The delivery of therapeutic nanoparticles to target cells is critical to their effectiveness. Here we quantified the impact of biological barriers on the delivery of nanoparticles to macrophages in two different tissues. We compared the delivery of gold nanoparticles to macrophages in the liver versus those in the tumor. We found that nanoparticle delivery to macrophages in the tumor was 75% less than to macrophages in the liver due to structural barriers. The tumor-associated macrophages took up more nanoparticles than Kupffer cells in the absence of barriers. Our results highlight the impact of biological barriers on nanoparticle delivery to cellular targets.

Currently, the limited capacity of pediatric acute myeloid leukemia (AML) therapies to prevent recurrence has contributed to high mortality rates. While dormant self-renewing leukemia stem cells (LSCs) contribute to adult AML relapse, their role in pediatric AML therapeutic resistance has not been clearly elucidated and thus was investigated in the context of this study. Through whole transcriptome sequencing (RNA-seq) analyses of FACS-purified human hematopoietic stem cells (HSCs; CD34 +CD38 -Lineage -) and progenitor cells (HPCs; CD34 +CD38 + Lineage -) from pediatric AML (n=10) compared with adult de novo (n=5) and secondary AML (n=6) as well as non-leukemic pediatric bone marrow samples (n=6), we identified widespread splicing alterations in pediatric AML compared to non-leukemic donors, indicative of a disruption in splicing regulation. In this study, we identified 2,000 exon skipping events in pediatric AML HSCs and HPCs. Moreover, we detected increased exon skipping and intron retention in stem cell self-renewal and survival transcripts in pediatric AML stem and progenitor cells. Specifically, the pro-survival isoform of MCL1, MCL1 long, was significantly increased in comparison to its pro-apoptotic counterpart, MCL1 short. In addition, self-renewal, RNA editing and splice isoform altering adenosine deaminase RNA specific 1 (ADAR1) p150 isoform levels were significantly (p=0.05) upregulated in pediatric AML progenitors suggesting that splicing and RNA editing deregulation could fuel pediatric AML stem and progenitor cell propagation. After successful completion of pre-IND development of a pharmacologically stable, potent, and selective small molecule splicing modulator, Rebecsinib (17S-FD-895) (Crews, Balain et al Cell Stem Cell 2016; Chan et al Cell Reports 2020), we developed a dual fluorescence lentiviral splicing reporter that assays the on target anti-leukemic efficacy of Rebcsinib and to assess the therapeutic index between LSCs and normal hematopoietic stem and progenitor cells. In hematopoietic progenitor assays, we observed a dose-dependent reduction in clonogenicity and replating of CD34 + cells isolated from pediatric AML samples following treatment with Rebecsinib. While pediatric AML samples were more sensitive to splicing modulation than adult de novo or adult secondary AML samples, normal cord blood progenitor samples were unaffected by splicing modulator treatment. In addition, we identified dose-dependent alterations in lentiviral splicing reporter activity in pediatric leukemia cells engrafted in a humanized AML mouse xenograft model following intravenous treatment with one dose of 10mg/kg and 20mg/kg of Rebecsinib. Finally, we observed a reduction in ADAR1 p150 transcripts by RNA-seq analysis of hematopoietic tissues in serially transplanted patient derived AML xenografts after Rebecsinib treatment suggesting that inhibition of ADAR1 splicing prevents LSC self-renewal. Cumulatively, these data demonstrate that stem and progenitor cell specific deregulation of pre-mRNA splicing and ADAR1 activation represent a therapeutic vulnerability to splicing modulation, which provides a strong rationale for developing Rebecsinib for preventing pediatric AML recurrence. Disclosures Cloos: Astellas: Speakers Bureau; DC-One: Other, Research Funding; Genentech: Research Funding; Helsinn: Other; Janssen: Research Funding; Merus: Other, Research Funding; Navigate: Patents & Royalties; Novartis: Consultancy, Other, Research Funding; Takeda: Research Funding. Crews: Ionis Pharmaceuticals: Research Funding. Burkart: Algenesis: Other: Co-founder. Jamieson: Forty Seven Inc.: Patents & Royalties.

Nanoparticles are promising vehicles for the precise delivery of molecular therapies to diseased sites. Nanoparticles interact with a series of tissues and cells before they reach their target, which causes less than 1% of administered nanoparticles to be delivered to these target sites. Researchers have been studying the nano-bio interactions that mediate nanoparticle delivery to develop guidelines for designing nanoparticles with enhanced delivery properties. In this review article, we describe these nano-bio interactions with a series of mathematical equations that quantitatively define the nanoparticle delivery process. We employ a compartment model framework to describe delivery where nanoparticles are either (1) at the site of administration, (2) in the vicinity of target cells, (3) internalized by the target cells, or (4) sequestered away in off-target sites or eliminated from the body. This framework explains how different biological processes govern nanoparticle transport between these compartments, and the role of intercompartmental transport rates in determining the final nanoparticle delivery efficiency. Our framework provides guiding principles to engineer nanoparticles for improved targeted delivery.

The surface of nanoparticles changes immediately after intravenous injection because blood proteins adsorb on the surface. How this interface changes during circulation and its impact on nanoparticle distribution within the body is not understood. Here, we developed a workflow to show that the evolution of proteins on nanoparticle surfaces predicts the biological fate of nanoparticles in vivo. This workflow involves extracting nanoparticles at multiple time points from circulation, isolating the proteins off the surface and performing proteomic mass spectrometry. The mass spectrometry protein library served as inputs, while blood clearance and organ accumulation were used as outputs to train a supervised deep neural network that predicts nanoparticle biological fate. In a double-blinded study, we tested the network by predicting nanoparticle spleen and liver accumulation with upward of 94% accuracy. Our neural network discovered that the mechanism of liver and spleen uptake is due to patterns of a multitude of nanoparticle surface adsorbed proteins. There are too many combinations to change these proteins manually using chemical or biological inhibitors to alter clearance. Therefore, we developed a technique that uses the host to act as a bioreactor to prepare nanoparticles with predictable clearance patterns that reduce liver and spleen uptake by 50% and 70%, respectively. These techniques provide opportunities to both predict nanoparticle behavior and also to engineer surface chemistries that are specifically designed by the body.

Studies into the interactions of serum proteins with nanoparticles are typically performed using nanoparticles that are larger than the size of proteins. Due to this size discrepancy, adsorbed proteins are commonly depicted as a globular structure surrounding a nanoparticle. Here, we asked how we should view nanoparticle–protein complexes when the nanoparticles are of similar size or smaller than the proteins with which they interact. We showed that nanoparticles can serve as a cargo on a protein rather than as a carrier of the protein in a size-dependent manner. This can occur when nanoparticles are below 10 nm in diameter. We discovered that when the nanoparticle is a cargo on the protein, the binding of the protein to the receptor target is minimally affected in contrast to the nanoparticle serving as a carrier. Our study should change how we view and describe nanoparticle–protein complexes when the nanoparticles involved are equal in size or smaller than proteins.

Patients infected with SARS‐CoV‐2 and influenza display similar symptoms, but treatment requirements are different. Clinicians need to accurately distinguish SARS‐CoV‐2 from influenza to provide appropriate treatment. Here, the authors develope a color‐based technique to differentiate between patients infected with SARS‐CoV‐2 and influenza A using a nucleic acid enzyme‐gold nanoparticle (GNP) molecular test requiring minimal equipment. The MNAzyme and GNP probes are designed to be robust to viral mutations. Conserved regions of the viral genomes are targeted, and two MNAzymes are created for each virus. The ability of the system to distinguish between SARS‐CoV‐2 and influenza A using 79 patient samples is tested. When detecting SARS‐CoV‐2 positive patients, the clinical sensitivity is 90%, and the specificity is 100%. When detecting influenza A, the clinical sensitivity and specificity are 93% and 100%, respectively. The high clinical performance of the MNAzyme‐GNP assay shows that it can be used to help clinicians choose effective treatments., Molecular diagnostic tests are needed to differentiate patients infected with COVID‐19 and influenza to guide treatment decisions. A colorimetric test using nucleic acid enzymes and gold nanoparticles is developed to identify these respiratory pathogens. The test has 93–95% sensitivity and 100% specificity in patients. The results support rapid translation of this materials‐based diagnostic approach for use in human patients.© 2021 WILEY‐VCH GmbHThis article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.

The field of nanotechnology has been a significant research focus in the last thirty years. This emphasis is due to the unique optical, electrical, magnetic, chemical and biological properties of materials approximately ten thousand times smaller than the diameter of a hair strand. Researchers have developed methods to synthesize and characterize large libraries of nanomaterials and have demonstrated their preclinical utility. We have entered a new phase of nanomedicine development, where the focus is to translate these technologies to benefit patients. This review article provides an overview of nanomedicine's unique properties, the current state of the field, and discusses the challenge of clinical translation. Finally, we discuss the need to build and strengthen partnerships between engineers and clinicians to create a feedback loop between the bench and bedside. This partnership will guide fundamental studies on the nanoparticle-biological interactions, address clinical challenges, and change the development and evaluation of new drug delivery systems, sensors, imaging agents, and therapeutic systems.

Limited tumor nanoparticle accumulation remains one of the main challenges in cancer nanomedicine. Here, we demonstrate that subtherapeutic photodynamic priming (PDP) enhances the accumulation of nanoparticles in subcutaneous murine prostate tumors ∼3-5-times without inducing cell death, vascular destruction, or tumor growth delay. We also found that PDP resulted in an ∼2-times decrease in tumor collagen content as well as a significant reduction of extracellular matrix density in the subendothelial zone. Enhanced nanoparticle accumulation combined with the reduced extravascular barriers improved therapeutic efficacy in the absence of off-target toxicity, wherein 5 mg/kg of Doxil with PDP was equally effective in delaying tumor growth as 15 mg/kg of Doxil. Overall, this study demonstrates the potential of PDP to enhance tumor nanomedicine accumulation and alleviate tumor desmoplasia without causing cell death or vascular destruction, highlighting the utility of PDP as a minimally invasive priming strategy that can improve therapeutic outcomes in desmoplastic tumors.

A nanoparticle can hold multiple types of therapeutic and imaging agents for disease treatment and diagnosis. However, controlling the storage of molecules in nanoparticles is challenging, because nonspecific intermolecular interactions are used for encapsulation. Here, we used specific DNA interactions to store molecules in nanoparticles. We made nanoparticles containing DNA anchors to capture DNA-conjugated small molecules. By changing the sequences and stoichiometry of DNA anchors, we can control the amount and ratio of molecules with different chemical properties in the nanoparticles. We modified the cytotoxicity of our nanoparticles to cancer cells by changing the ratio of encapsulated drugs (mertansine and doxorubicin). Specifically controlling the storage of multiple types of molecules allows us to optimize the properties of combination drug and imaging nanoparticles.

The delivery of medical agents to a specific diseased tissue or cell is critical for diagnosing and treating patients. Nanomaterials are promising vehicles to transport agents that include drugs, contrast agents, immunotherapies and gene editors. They can be engineered to have different physical and chemical properties that influence their interactions with their biological environments and delivery destinations. In this Review Article, we discuss nanoparticle delivery systems and how the biology of disease should inform their design. We propose developing a framework for building optimal delivery systems that uses nanoparticle–biological interaction data and computational analyses to guide future nanomaterial designs and delivery strategies. This Review proposes a framework for designing delivery systems to target diseased tissues based on the biology of the target, the journey and computational algorithms.

There is a tremendous focus on the application of nanomaterials for the treatment of cancer. Nonprimate models are conventionally used to assess the biomedical utility of nanomaterials. However, these animals often lack an intact immunological background, and the tumors in these animals do not develop spontaneously. We introduce a preclinical woodchuck hepatitis virus-induced liver cancer model as a platform for nanoparticle (NP)-based in vivo experiments. Liver cancer development in these out-bred animals occurs as a result of persistent viral infection, mimicking human hepatitis B virus-induced HCC development. We highlight how this model addresses key gaps associated with other commonly used tumor models. We employed this model to (1) track organ biodistribution of gold NPs after intravenous administration, (2) examine their subcellular localization in the liver, (3) determine clearance kinetics, and (4) characterize the identity of hepatic macrophages that take up NPs using RNA-sequencing (RNA-seq). We found that the liver and spleen were the primary sites of NP accumulation. Subcellular analyses revealed accumulation of NPs in the lysosomes of CD14+ cells. Through RNA-seq, we uncovered that immunosuppressive macrophages within the woodchuck liver are the major cell type that take up injected NPs. The woodchuck-HCC model has the potential to be an invaluable tool to examine NP-based immune modifiers that promote host anti-tumor immunity.

COVID-19 has spread globally since its discovery in Hubei province, China in December 2019. A combination of computed tomography imaging, whole genome sequencing, and electron microscopy were initially used to screen and identify SARS-CoV-2, the viral etiology of COVID-19. The aim of this review article is to inform the audience of diagnostic and surveillance technologies for SARS-CoV-2 and their performance characteristics. We describe point-of-care diagnostics that are on the horizon and encourage academics to advance their technologies beyond conception. Developing plug-and-play diagnostics to manage the SARS-CoV-2 outbreak would be useful in preventing future epidemics.

Three-dimensional (3D) optical microscopy can be used to understand and improve the delivery of nanomedicine. However, this approach cannot be performed for analyzing liposomes in tissues because the processing step to make tissues transparent for imaging typically removes the lipids. Here, we developed a tag, termed REMNANT, that enables 3D imaging of organic materials in biological tissues. We demonstrated the utility of this tag for the 3D mapping of liposomes in intact tissues. We also showed that the tag is able to monitor the release of entrapped therapeutic agents. We found that liposomes release their cargo >100-fold faster in tissues in vivo than in conventional in vitro assays. This allowed us to design a liposomal formulation with enhanced ability to kill tumor associated macrophages. Our development opens up new opportunities for studying the chemical properties and pharmacodynamics of administered organic materials in an intact biological environment. This approach provides insight into the in vivo behavior of degradable materials, where the newly discovered information can guide the engineering of the next generation of imaging and therapeutic agents.

Diagnostic tests can detect diseases, monitor responses, and inform treatments. They are vital to the effective management of disease. There have been significant advances in the engineering of new diagnostic technologies. These technologies may forgo sample extraction, simplify readout, or automate processing. Many researchers design these diagnostics based on test performance in a limited sample subset. This approach ignores the intertwined relationship between patient characteristics and diagnostic test results. Yet, it is important to understand the clinical decision-making workflow and how the disease manifests in order to optimally design diagnostic tests. This review article explores the three aspects of incorporating patient characteristics to maximize diagnostic performance. 1) Characterize patient populations using patient demographics, disease prevalence, and other unique features. 2) Use the characteristics of the patient population to establish design requirements. 3) Determine the best use case since each case has different performance and target requirements. In this framework the clinical, technological, and unmet needs of a patient population shape the diagnostics design requirements. Following these steps will lead to maximal diagnostic performance and poise new diagnostics for real world use.

Coating the nanoparticle surface with cancer cell recognizing ligands is expected to facilitate specific delivery of nanoparticles to diseased cells in vivo. While this targeting strategy is appealing, no nanoparticle-based active targeting formulation for solid tumor treatment had made it past phase III clinical trials. Here, we quantified the cancer cell-targeting efficiencies of Trastuzumab (Herceptin) and folic acid coated gold and silica nanoparticles in multiple mouse tumor models. Surprisingly, we showed that less than 14 out of 1 million (0.0014% injected dose) intravenously administrated nanoparticles were delivered to targeted cancer cells, and that only 2 out of 100 cancer cells interacted with the nanoparticles. The majority of the intratumoral nanoparticles were either trapped in the extracellular matrix or taken up by perivascular tumor associated macrophages. The low cancer cell targeting efficiency and significant uptake by noncancer cells suggest the need to re-evaluate the active targeting process and therapeutic mechanisms using quantitative methods. This will be important for developing strategies to deliver emerging therapeutics such as genome editing, nucleic acid therapy, and immunotherapy for cancer treatment using nanocarriers.

Medical diagnostic assays provide exquisite sensitivity and precision in the diagnoses of patients. However, these technologies often require multiple steps, skilled technicians, and facilities to store heat-sensitive reagents. Here, we developed a high-throughput compression method to incorporate different assay components into color-coded tablets. With our technique, premeasured quantities of reagents can be encapsulated in compressed tablets. We show that tableting stabilizes heat-sensitive reagents and simplifies a broad range of assays, including isothermal nucleic acid amplification techniques, enzyme-based immunoassays, and microbead diagnostics. To test the clinical readiness of this tableting technology, we show the ability of tableted diagnostics for screening hepatitis B-positive patient samples. Our development simplifies complicated assays and the transportation of reagents and mitigates the need for refrigeration of reagents. This advances the use of complex assays in remote areas with limited infrastructure.

Rapid, simple, and cost-effective diagnostics are needed to improve healthcare at the point of care (POC). However, the most widely used POC diagnostic, the lateral flow immunoassay (LFA), is ~1000-times less sensitive and has a smaller analytical range than laboratory tests, requiring a confirmatory test to establish truly negative results. Here, a rational and systematic strategy is used to design the LFA contrast label (i.e., gold nanoparticles) to improve the analytical sensitivity, analytical detection range, and antigen quantification of LFAs. Specifically, we discovered that the size (30, 60, or 100 nm) of the gold nanoparticles is a main contributor to the LFA analytical performance through both the degree of receptor interaction and the ultimate visual or thermal contrast signals. Using the optimal LFA design, we demonstrated the ability to improve the analytical sensitivity by 256-fold and expand the analytical detection range from 3 log10 to 6 log10 for diagnosing patients with inflammatory conditions by measuring C-reactive protein. This work demonstrates that, with appropriate design of the contrast label, a simple and commonly used diagnostic technology can compete with more expensive state-of-the-art laboratory tests.

Infectious diseases are a major global threat that accounts for one of the leading causes of global mortality and morbidity. Prompt diagnosis is a crucial first step in the management of infectious threats, which aims to quarantine infected patients to avoid contacts with healthy individuals and deliver effective treatments prior to further spread of diseases. This review article discusses current advances of diagnostic systems using colloidal nanomaterials (e.g., gold nanoparticles, quantum dots, magnetic nanoparticles) for identifying and differentiating infectious pathogens. The challenges involved in the clinical translation of these emerging nanotechnology based diagnostic devices will also be discussed.

Since its discovery in 1977, the study of alternative RNA splicing has revealed a plethora of mechanisms that had never before been documented in nature. Understanding these transitions and their outcome at the level of the cell and organism has become one of the great frontiers of modern chemical biology. Until 2007, this field remained in the hands of RNA biologists. However, the recent identification of natural product and synthetic modulators of RNA splicing has opened new access to this field, allowing for the first time a chemical-based interrogation of RNA splicing processes. Simultaneously, we have begun to understand the vital importance of splicing in disease, which offers a new platform for molecular discovery and therapy. As with many natural systems, gaining clear mechanistic detail at the molecular level is key towards understanding the operation of any biological machine. This minireview presents recent lessons learned in this emerging field of RNA splicing chemistry and chemical biology.

Chemical probes are key components of the bioimaging toolbox, as they label biomolecules in cells and tissues. The new challenge in bioimaging is to design chemical probes for three-dimensional (3D) tissue imaging. In this work, we discovered that light scattering of metal nanoparticles can provide 3D imaging contrast in intact and transparent tissues. The nanoparticles can act as a template for the chemical growth of a metal layer to further enhance the scattering signal. The use of chemically grown nanoparticles in whole tissues can amplify the scattering to produce a 1.4 million-fold greater photon yield than obtained using common fluorophores. These probes are non-photobleaching and can be used alongside fluorophores without interference. We demonstrated three distinct biomedical applications: (a) molecular imaging of blood vessels, (b) tracking of nanodrug carriers in tumors, and (c) mapping of lesions and immune cells in a multiple sclerosis mouse model. Our strategy establishes a distinct yet complementary set of imaging probes for understanding disease mechanisms in three dimensions.

We have shown that nanoparticles (NPs) can be used as ligand-multimerization platforms to activate specific cellular receptors in vivo. Nanoparticles coated with autoimmune disease-relevant peptide-major histocompatibility complexes (pMHC) blunted autoimmune responses by triggering the differentiation and expansion of antigen-specific regulatory T cells in vivo. Here, we define the engineering principles impacting biological activity, detail a synthesis process yielding safe and stable compounds, and visualize how these nanomedicines interact with cognate T cells. We find that the triggering properties of pMHC-NPs are a function of pMHC intermolecular distance and involve the sustained assembly of large antigen receptor microclusters on murine and human cognate T cells. These compounds show no off-target toxicity in zebrafish embryos, do not cause haematological, biochemical or histological abnormalities, and are rapidly captured by phagocytes or processed by the hepatobiliary system. This work lays the groundwork for the design of ligand-based NP formulations to re-program in vivo cellular responses using nanotechnology.

A significant challenge to delivering therapeutic doses of nanoparticles to targeted disease sites is the fact that most nanoparticles become trapped in the liver. Liver-resident macrophages, or Kupffer cells, are key cells in the hepatic sequestration of nanoparticles. However, the precise role that the macrophage phenotype plays in nanoparticle uptake is unknown. Here, we show that the human macrophage phenotype modulates hard nanoparticle uptake. Using gold nanoparticles, we examined uptake by human monocyte-derived macrophages that had been driven to a "regulatory" M2 phenotype or an "inflammatory" M1 phenotype and found that M2-type macrophages preferentially take up nanoparticles, with a clear hierarchy among the subtypes (M2c > M2 > M2a > M2b > M1). We also found that stimuli such as LPS/IFN-γ rather than with more "regulatory" stimuli such as TGF-β/IL-10 reduce per cell macrophage nanoparticle uptake by an average of 40%. Primary human Kupffer cells were found to display heterogeneous expression of M1 and M2 markers, and Kupffer cells expressing higher levels of M2 markers (CD163) take up significantly more nanoparticles than Kupffer cells expressing lower levels of surface CD163. Our results demonstrate that hepatic inflammatory microenvironments should be considered when studying liver sequestration of nanoparticles, and that modifying the hepatic microenvironment might offer a tool for enhancing or decreasing this sequestration. Our findings also suggest that models examining the nanoparticle/macrophage interaction should include studies with primary tissue macrophages.

[Image: see text] The spliceosome has been shown to be a promising target for the development of new anticancer therapeutics. Synthetic and chemical biological efforts directed toward the development of natural product-based splice modulators (SPLMs) have shown that the potency of these compounds derives from their ability to selectively affect the alternate splicing of apoptotic genes in tumor cells. However, questions remain regarding the mechanistic understanding of splice modulation as well as the selectivity with which SPLMs impact certain genes.

Pancreatic islets are fragile cell clusters and many isolated islets are not suitable for transplantation. Furthermore, following transplantation, islets will experience a state of hypoxia and poor nutrient diffusion before revascularization, which is detrimental to islet survival; this is affected by islet size and health. Here we engineered tuneable size-controlled pseudo-islets created by dispersing de-aggregated islets in an endothelialized collagen scaffold. This supported subcutaneous engraftment, which returned streptozotocin-induced diabetic mice to normoglycemia. Whole-implant imaging after tissue clearing demonstrated pseudo-islets regenerated their vascular architecture and insulin-secreting β-cells were within 5 μm of a perfusable vessel – a feature unique to this approach. By using an endothelialized collagen scaffold, this work highlights a novel “bottom-up” approach to islet engineering that provides control over the size and composition of the constructs, while enabling the critical ability to revascularize and engraft when transplanted into the clinically useful subcutaneous space.

Lymph node follicles capture and retain antigens to induce germinal centers and long-lived humoral immunity. However, control over antigen retention has been limited. Here we discovered that antigen conjugated to nanoparticle carriers of different sizes impacts the intralymph node transport and specific cell interaction. We found that follicular dendritic cell (FDC) networks determine the intralymph node follicle fate of these nanoparticles by clearing smaller ones (5-15 nm) within 48 h and retaining larger ones (50-100 nm) for over 5 weeks. The 50-100 nm-sized nanoparticles had 175-fold more delivery of antigen at the FDC dendrites, 5-fold enhanced humoral immune responses of germinal center B cell formation, and 5-fold more antigen-specific antibody production over 5-15 nm nanoparticles. Our results show that we can tune humoral immunity by simply manipulating the carrier size design to produce effectiveness of vaccines.

Mobile phone technology is a perfect companion for point-of-care diagnostics as they come equipped with advanced processors, high resolution cameras, and network connectivity. Despite several academic pursuits, only a few mobile phone diagnostics have been tested in the field, commercialized or achieved regulatory approval. This review will address the challenges associated with developing mobile diagnostics and suggest strategies to overcome them. We aim to provide a resource for researchers to accelerate the development of new diagnostics. Our Account includes an overview of published mobile phone diagnostics and highlights lessons learned from their approach to diagnostic development. Also, we have included recommendations from regulatory and public health agencies, such as the U.S. Food and Drug Administration and World Health Organization, to further guide researchers. We believe that the development of mobile phone point-of-care diagnostics takes place in four distinct steps: (1) Needs and Value Assessment, (2) Technology Development, (3) Preclinical Verification, and (4) Clinical Validation and Field Trials. During each step, we outline developmental strategies to help researchers avoid potential challenges. (1) Researchers commonly develop devices to maximize technical parameters such as sensitivity and time which do not necessarily translate to increased clinical impact. Researchers must focus on assessing specific diagnostic needs and the value which a potential device would offer. (2) Often, researchers claim they have developed devices for feasible implementation at the point-of-care, yet they rely on laboratory resources. Researchers must develop equipment-free devices which are agnostic to any mobile phone. (3) Another challenge researchers face is decreased performance during field evaluations relative to initial laboratory verification. Researchers must ensure that they simulate the field conditions during laboratory verification to achieve successful translation. (4) Finally, proper field testing of devices must be performed in conditions which match that of the final intended use. The future of mobile phone point-of-care diagnostic devices is bright and has the potential to radically change how patients are diagnosed. Before we reach this point, researchers must take a step backward and focus on the first-principles of basic research. The widespread adoption and rapid scaling of these devices can only be achieved once the fundamentals have been considered. The insights and strategies provided here will help researchers avoid pitfalls, streamline development and make better decisions during the development of new diagnostics. Further, we believe this Account can help push the field of mobile diagnostics toward increased productivity, leading to more approved devices and ultimately helping curb the burden of disease worldwide.

We describe the design, synthesis, and antitumor activity of an 18 carbon α,ω-dicarboxylic acid monoconjugated via an ester linkage to paclitaxel (PTX). This 1,18-octadecanedioic acid-PTX (ODDA-PTX) prodrug readily forms a noncovalent complex with human serum albumin (HSA). Preservation of the terminal carboxylic acid moiety on ODDA-PTX enables binding to HSA in the same manner as native long-chain fatty acids (LCFAs), within hydrophobic pockets, maintaining favorable electrostatic contacts between the ω-carboxylate of ODDA-PTX and positively charged amino acid residues of the protein. This carrier strategy for small molecule drugs is based on naturally evolved interactions between LCFAs and HSA, demonstrated here for PTX. ODDA-PTX shows differentiated pharmacokinetics, higher maximum tolerated doses and increased efficacy in vivo in multiple subcutaneous murine xenograft models of human cancer, as compared to two FDA-approved clinical formulations, Cremophor EL-formulated paclitaxel (crPTX) and Abraxane (nanoparticle albumin-bound (nab)-paclitaxel).

Metastasis of solid tumors is a key determinant of cancer patient survival. Targeting micrometastases using nanoparticles could offer a way to stop metastatic tumor growth before it causes excessive patient morbidity. However, nanoparticle delivery to micrometastases is difficult to investigate because micrometastases are small in size and lie deep within tissues. Here, we developed an imaging and image analysis workflow to analyze nanoparticle–cell interactions in metastatic tumors. This technique combines tissue clearing and 3D microscopy with machine learning-based image analysis to assess the physiology of micrometastases with single-cell resolution and quantify the delivery of nanoparticles within them. We show that nanoparticles access a higher proportion of cells in micrometastases (50% nanoparticle-positive cells) compared with primary tumors (17% nanoparticle-positive cells) because they reside close to blood vessels and require a small diffusion distance to reach all tumor cells. Furthermore, the high-throughput nature of our image analysis workflow allowed us to profile the physiology and nanoparticle delivery of 1,301 micrometastases. This enabled us to use machine learning-based modeling to predict nanoparticle delivery to individual micrometastases based on their physiology. Our imaging method allows researchers to measure nanoparticle delivery to micrometastases and highlights an opportunity to target micrometastases with nanoparticles. The development of models to predict nanoparticle delivery based on micrometastasis physiology could enable personalized treatments based on the specific physiology of a patient’s micrometastases.