Engineering a Rigid Nucleic Acid Structure to Improve the Limit of Detection for Genetic Assays.

Detecting nucleic acids at ultralow concentrations is critical for research and clinical applications. Particle-based assays are commonly used to detect nucleic acids. However, DNA hybridization on particle surfaces is inefficient due to the instability of tethered sequences, which negatively influences the assay's detection sensitivity. Here, we report a method to stabilize sequences on particle surfaces using a double-stranded linker at the 5' end of the tethered sequence. We termed this method Rigid Double Stranded Genomic Linkers for Improved DNA Analysis (RIGID-DNA). Our method led to a 3- and 100-fold improvement of the assays' clinical and analytical sensitivity, respectively. Our approach can enhance the hybridization efficiency of particle-based assays without altering existing assay workflows. This approach can be adapted to other platforms and surfaces to enhance the detection sensitivity.

The exit of nanoparticles from solid tumours.

Nanoparticles enter tumours through endothelial cells, gaps or other mechanisms, but how they exit is unclear. The current paradigm states that collapsed tumour lymphatic vessels impair the exit of nanoparticles and lead to enhanced retention. Here we show that nanoparticles exit the tumour through the lymphatic vessels within or surrounding the tumour. The dominant lymphatic exit mechanism depends on the nanoparticle size. Nanoparticles that exit the tumour through the lymphatics are returned to the blood system, allowing them to recirculate and interact with the tumour in another pass. Our results enable us to define a mechanism of nanoparticle delivery to solid tumours alternative to the enhanced permeability and retention effect. We call this mechanism the active transport and retention principle. This delivery principle provides a new framework to engineer nanomedicines for cancer treatment and detection.

Identifying cell receptors for the nanoparticle protein corona using genome screens.

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 Bind to Endothelial Cells in Injured Blood Vessels via a Transient Protein Corona.

Nanoparticles travel through blood vessels to reach disease sites, but the local environment they encounter may affect their surface chemistry and cellular interactions. Here, we found that as nanoparticles transit through injured blood vessels they may interact with a highly localized concentration of platelet factor 4 proteins released from activated platelets. The platelet factor 4 binds to the nanoparticle surface and interacts with heparan sulfate proteoglycans on endothelial cells, and induces uptake. Understanding nanoparticle interactions with blood proteins and endothelial cells during circulation is critical to optimizing their design for diseased tissue targeting and delivery.

Gas Vesicle-Blood Interactions Enhance Ultrasound Imaging Contrast.

Gas vesicles (GVs) are genetically encoded, air-filled protein nanostructures of broad interest for biomedical research and clinical applications, acting as imaging and therapeutic agents for ultrasound, magnetic resonance, and optical techniques. However, the biomedical applications of GVs as systemically injectable nanomaterials have been hindered by a lack of understanding of GVs' interactions with blood components, which can significantly impact in vivo behavior. Here, we investigate the dynamics of GVs in the bloodstream using a combination of ultrasound and optical imaging, surface functionalization, flow cytometry, and mass spectrometry. We find that erythrocytes and serum proteins bind to GVs and shape their acoustic response, circulation time, and immunogenicity. We show that by modifying the GV surface we can alter these interactions and thereby modify GVs' in vivo performance. These results provide critical insights for the development of GVs as agents for nanomedicine.

Toward Predicting Nanoparticle Distribution in Heterogeneous Tumor Tissues.

Nanobio interaction studies have generated a significant amount of data. An important next step is to organize the data and design computational techniques to analyze the nanobio interactions. Here we developed a computational technique to correlate the nanoparticle spatial distribution within heterogeneous solid tumors. This approach led to greater than 88% predictive accuracy of nanoparticle location within a tumor tissue. This proof-of-concept study shows that tumor heterogeneity might be defined computationally by the patterns of biological structures within the tissue, enabling the identification of tumor patterns for nanoparticle accumulation.

Genotyping SARS-CoV-2 Variants Using Ratiometric Nucleic Acid Barcode Panels.

Designing diagnostic assays to genotype rapidly mutating viruses remains a challenge despite the overall improvements in nucleic acid detection technologies. RT-PCR and next-generation sequencing are unsuitable for genotyping during outbreaks or in point-of-care detection due to their infrastructure requirements and longer turnaround times. We developed a quantum dot barcode multiplexing system to genotype mutated viruses. We designed multiple quantum dot barcodes to target conserved, wildtype, and mutated regions of SARS-CoV-2. We calculated ratios of the signal output from different barcodes that enabled SARS-CoV-2 detection and identified SARS-CoV-2 variant strains from a sample. We detected different sequence types, including conserved genes, nucleotide deletions, and single nucleotide substitutions. Our system detected SARS-CoV-2 patient specimens with 98% sensitivity and 94% specificity across 91 patient samples. Further, we leveraged our barcoding and ratio system to track the emergence of the N501Y SARS-CoV-2 mutation from December 2020 to May 2021 and demonstrated that the more transmissible N501Y mutation started to dominate infections by April 2021. Our barcoding and signal ratio approach can genotype viruses and track the emergence of viral mutations in a single diagnostic test. This technology can be extended to tracking other viruses. Combined with smartphone detection technologies, this assay can be adapted for point-of-care tracking of viral mutations in real time.

Tunable and precise miniature lithium heater for point-of-care applications.

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 (∆TRT = 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.

Sequential Reagent Release from a Layered Tablet for Multistep Diagnostic Assays.

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 (tpp47 gene from Treponema pallidum) and nitrite ions in an aqueous sample without user intervention. Our multilayer tablets can simplify multistep assay processes.

Impact of Tumor Barriers on Nanoparticle Delivery to Macrophages.

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.

Assessing micrometastases as a target for nanoparticles using 3D microscopy and machine learning.

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.

Subtherapeutic Photodynamic Treatment Facilitates Tumor Nanomedicine Delivery and Overcomes Desmoplasia.

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.

Surveilling and Tracking COVID-19 Patients Using a Portable Quantum Dot Smartphone Device.

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.

Nanotechnology for modern medicine: next step towards clinical translation.

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.

The dose threshold for nanoparticle tumour delivery.

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.

The entry of nanoparticles into solid tumours.

The concept of nanoparticle transport through gaps between endothelial cells (inter-endothelial gaps) in the tumour blood vessel is a central paradigm in cancer nanomedicine. The size of these gaps was found to be up to 2,000 nm. This justified the development of nanoparticles to treat solid tumours as their size is small enough to extravasate and access the tumour microenvironment. Here we show that these inter-endothelial gaps are not responsible for the transport of nanoparticles into solid tumours. Instead, we found that up to 97% of nanoparticles enter tumours using an active process through endothelial cells. This result is derived from analysis of four different mouse models, three different types of human tumours, mathematical simulation and modelling, and two different types of imaging techniques. These results challenge our current rationale for developing cancer nanomedicine and suggest that understanding these active pathways will unlock strategies to enhance tumour accumulation.

Liposome Imaging in Optically Cleared Tissues.

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.

An Analysis of the Binding Function and Structural Organization of the Protein Corona.

Blood proteins adsorb onto the surface of nanoparticles after intravenous injection to form a protein corona. The underlying organization and binding function of these adsorbed proteins remain unclear. This can impact how the corona mediates cell and tissue interactions. Here, we investigated the function and structural organization of the protein corona using an immunoassay approach. We discovered that only 27% of the adsorbed proteins examined are functional for binding to their target protein. This is because the corona architecture is not a monolayer, but an assembly of proteins that are bound to each other. We further demonstrated that we can control the binding functionality of a protein by changing the organization of proteins in the assembly. We show that manipulation of the corona protein composition and assembly can influence their interactions with macrophage cells in culture. This study provides detailed functional and structural insights into the protein corona on nanomaterials and offers a new strategy to manipulate it for controlled interactions with the biological system.

DNA-Controlled Encapsulation of Small Molecules in Protein Nanoparticles.

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.

Engineering Steps for Mobile Point-of-Care Diagnostic Devices.

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.