Mechanisms and Barriers in Nanomedicine: Progress in the Field and Future Directions.

In recent years, steady progress has been made in synthesizing and characterizing engineered nanoparticles, resulting in several approved drugs and multiple promising candidates in clinical trials. Regulatory agencies such as the Food and Drug Administration and the European Medicines Agency released important guidance documents facilitating nanoparticle-based drug product development, particularly in the context of liposomes and lipid-based carriers. Even with the progress achieved, it is clear that many barriers must still be overcome to accelerate translation into the clinic. At the recent conference workshop "Mechanisms and Barriers in Nanomedicine" in May 2023 in Colorado, U.S.A., leading experts discussed the formulation, physiological, immunological, regulatory, clinical, and educational barriers. This position paper invites open, unrestricted, nonproprietary discussion among senior faculty, young investigators, and students to trigger ideas and concepts to move the field forward.

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.

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.

Principles of Nanoparticle Delivery to Solid Tumors.

The effective treatment of patients with cancer hinges on the delivery of therapeutics to a tumor site. Nanoparticles provide an essential transport system. We present 5 principles to consider when designing nanoparticles for cancer targeting: (a) Nanoparticles acquire biological identity in vivo, (b) organs compete for nanoparticles in circulation, (c) nanoparticles must enter solid tumors to target tumor components, (d) nanoparticles must navigate the tumor microenvironment for cellular or organelle targeting, and (e) size, shape, surface chemistry, and other physicochemical properties of nanoparticles influence their transport process to the target. This review article describes these principles and their application for engineering nanoparticle delivery systems to carry therapeutics to tumors or other disease targets.

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.

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 a systemically injectable nanomaterial have been hindered by a lack of understanding of GVs' interactions with blood components, which can significantly impact in vivo performance. 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.

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.

Delineating the tumour microenvironment response to a lipid nanoparticle formulation.

Nanoparticles can reduce cytotoxicity, increase circulation time and increase accumulation in tumours compared to free drug. However, the value of using nanoparticles for carrying small molecules to treat tumours at the cellular level has been poorly established. Here we conducted a cytodistribution analysis on Doxorubicin-treated and Doxil-treated tumours to delineate the differences between the small molecule therapeutic Doxorubicin and its packaged liposomal formulation Doxil. We found that Doxil kills more cancer cells, macrophages and neutrophils in the 4T1 breast cancer tumour model, but there is delayed killing compared to its small molecule counterpart Doxorubicin. The cellular interaction with Doxil has slower uptake kinetics and the particles must be degraded to release the drug and kill the cells. We also found that macrophages and neutrophils in Doxil-treated tumours repopulated faster than cancer cells during the relapse phase. While researchers conventionally use tumour volume and animal survival to determine a therapeutic effect, our results show diverse cell killing and a greater amount of cell death in vivo after Doxil liposomes are administered. We conclude that the fate and behaviour of the nanocarrier influences its effectiveness as a cancer therapy. Further investigations on the interactions between different nanoparticle designs and the tumour microenvironment components will lead to more precise engineering of nanocarriers to selectively kill tumour cells and prolong the therapeutic effect.

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.

A proposed mathematical description of in vivo nanoparticle delivery.

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.

Gold Nanoparticle Smartphone Platform for Diagnosing Urinary Tract Infections.

Current urinary tract infection (UTI) diagnostic methods are slow or provide limited information, resulting in prescribing antibiotic therapy before bacterial pathogen identification. Here, we adapted a gold nanoparticle colorimetric approach and developed a smartphone platform for UTI detection. We show the parallel identification of five major UTI pathogens at clinically relevant concentrations of 105 bacteria/mL using bacteria-specific and universal probes. We validated the diagnostic technology using 115 positive and 19 negative samples from patients with Escherichia coli, Proteus mirabilis, and Klebsiella pneumoniae infections. The assay successfully identified the infecting pathogen (specificity: >98% and sensitivity: 51-73%) in 3 h. Our platform is faster than culturing and can wirelessly store and transmit results at the cost of $0.38 per assay.

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.

Why nanoparticles prefer liver macrophage cell uptake in vivo.

Effective delivery of therapeutic and diagnostic nanoparticles is dependent on their ability to accumulate in diseased tissues. However, most nanoparticles end up in liver macrophages regardless of nanoparticle design after administration. In this review, we describe the interactions of liver macrophages with nanoparticles. Liver macrophages have significant advantages in interacting with circulating nanoparticles over most target cells and tissues in the body. We describe these advantages in this article. Understanding these advantages will enable the development of strategies to overcome liver macrophages and deliver nanoparticles to targeted diseased tissues effectively. Ultimately, these approaches will increase the therapeutic efficacy and diagnostic signal of nanoparticles.