Deep learning-assisted sensitive detection of fentanyl using a bubbling-microchip.

Deep learning-enabled smartphone-based image processing has significant advantages in the development of point-of-care diagnostics. Conventionally, most deep-learning applications require task specific large scale expertly annotated datasets. Therefore, these algorithms are oftentimes limited only to applications that have large retrospective datasets available for network development. Here, we report the possibility of utilizing adversarial neural networks to overcome this challenge by expanding the utility of non-specific data for the development of deep learning models. As a clinical model, we report the detection of fentanyl, a small molecular weight drug that is a type of opioid, at the point-of-care using a deep-learning empowered smartphone assay. We used the catalytic property of platinum nanoparticles (PtNPs) in a smartphone-enabled microchip bubbling assay to achieve high analytical sensitivity (detecting fentanyl at concentrations as low as 0.23 ng mL-1 in phosphate buffered saline (PBS), 0.43 ng mL-1 in human serum and 0.64 ng mL-1 in artificial human urine). Image-based inferences were made by our adversarial-based SPyDERMAN network that was developed using a limited dataset of 104 smartphone images of microchips with bubble signals from tests performed with known fentanyl concentrations and using our retrospective library of 17 573 non-specific bubbling-microchip images. The accuracy (± standard error of mean) of the developed system in determining the presence of fentanyl, when using a cutoff concentration of 1 ng mL-1, was 93 ± 0% in human serum (n = 100) and 95.3 ± 1.5% in artificial human urine (n = 100).

Macrophage-Encapsulated Bioorthogonal Nanozymes for Targeting Cancer Cells.

Macrophages migrate to tumor sites by following chemoattractant gradients secreted by tumor cells, providing a truly active targeting strategy for cancer therapy. However, macrophage-based delivery faces challenges of cargo loading, control of release, and effects of the payload on the macrophage vehicle. We present a strategy that employs bioorthogonal "nanozymes" featuring transition metal catalysts (TMCs) to provide intracellular "factories" for the conversion of prodyes and prodrugs into imaging agents and chemotherapeutics. These nanozymes solubilize and stabilize the TMCs by embedding them into self-assembled monolayer coating gold nanoparticles. Nanozymes delivered into macrophages were intracellularly localized and retained activity even after prolonged (72 h) incubation. Significantly, nanozyme-loaded macrophages maintained their inherent migratory ability toward tumor cell chemoattractants, efficiently killing cancer cells in cocultures. This work establishes the potential of nanozyme-loaded macrophages for tumor site activation of prodrugs, providing readily tunable dosages and delivery rates while minimizing off-target toxicity of chemotherapeutics.

Selective treatment of intracellular bacterial infections using host cell-targeted bioorthogonal nanozymes.

Intracellular bacterial infections are difficult to treat, and in the case of Salmonella and related infections, can be life threatening. Antibiotic treatments for intracellular infections face challenges including cell penetration and intracellular degradation that both reduce antibiotic efficacy. Even when treatable, the increased dose of antibiotics required to counter infections can strongly impact the microbiome, compromising the native roles of beneficial non-pathogenic species. Bioorthogonal catalysis provides a new tool to combat intracellular infections. Catalysts embedded in the monolayers of gold nanoparticles (nanozymes) bioorthogonally convert inert antibiotic prodrugs (pro-antibiotics) into active species within resident macrophages. Targeted nanozyme delivery to macrophages was achieved through mannose conjugation and subsequent uptake VIA the mannose receptor (CD206). These nanozymes efficiently converted pro-ciprofloxacin to ciprofloxacin inside the macrophages, selectively killing pathogenic Salmonella enterica subsp. enterica serovar Typhimurium relative to non-pathogenic Lactobacillus sp. in a transwell co-culture model. Overall, this targeted bioorthogonal nanozyme strategy presents an effective treatment for intracellular infections, including typhoid and tuberculosis.

Could artificial intelligence revolutionize the development of nanovectors for gene therapy and mRNA vaccines?

Gene therapy enables the introduction of nucleic acids like DNA and RNA into host cells, and is expected to revolutionize the treatment of a wide range of diseases. This growth has been further accelerated by the discovery of CRISPR/Cas technology, which allows accurate genomic editing in a broad range of cells and organisms in vitro and in vivo. Despite many advances in gene delivery and the development of various viral and non-viral gene delivery vectors, the lack of highly efficient non-viral systems with low cellular toxicity remains a challenge. The application of cutting-edge technologies such as artificial intelligence (AI) has great potential to find new paradigms to solve this issue. Herein, we review AI and its major subfields including machine learning (ML), neural networks (NNs), expert systems, deep learning (DL), computer vision and robotics. We discuss the potential of AI-based models and algorithms in the design of targeted gene delivery vehicles capable of crossing extracellular and intracellular barriers by viral mimicry strategies. We finally discuss the role of AI in improving the function of CRISPR/Cas systems, developing novel nanobots, and mRNA vaccine carriers.

SARS-CoV-2 RNA Detection by a Cellphone-Based Amplification-Free System with CRISPR/CAS-Dependent Enzymatic (CASCADE) Assay.

CRISPR (Clustered regularly interspaced short palindromic repeats)-based diagnostic technologies have emerged as a promising alternative to accelerate delivery of SARS-CoV-2 molecular detection at the point of need. However, efficient translation of CRISPR-diagnostic technologies to field application is still hampered by dependence on target amplification and by reliance on fluorescence-based results readout. Herein, an amplification-free CRISPR/Cas12a-based diagnostic technology for SARS-CoV-2 RNA detection is presented using a smartphone camera for results readout. This method, termed Cellphone-based amplification-free system with CRISPR/CAS-dependent enzymatic (CASCADE) assay, relies on mobile phone imaging of a catalase-generated gas bubble signal within a microfluidic channel and does not require any external hardware optical attachments. Upon specific detection of a SARS-CoV-2 reverse-transcribed DNA/RNA heteroduplex target (orf1ab) by the ribonucleoprotein complex, the transcleavage collateral activity of the Cas12a protein on a Catalase:ssDNA probe triggers the bubble signal on the system. High analytical sensitivity in signal detection without previous target amplification (down to 50 copies µL-1) is observed in spiked samples, in ≈71 min from sample input to results readout. With the aid of a smartphone vision tool, high accuracy (AUC = 1.0; CI: 0.715 - 1.00) is achieved when the CASCADE system is tested with nasopharyngeal swab samples of PCR-positive COVID-19 patients.

Nanodelivery vehicles induce remote biochemical changes in vivo.

Nanomaterial-based platforms are promising vehicles for the controlled delivery of therapeutics. For these systems to be both efficacious and safe, it is essential to understand where the carriers accumulate and to reveal the site-specific biochemical effects they produce in vivo. Here, a dual-mode mass spectrometry imaging (MSI) method is used to evaluate the distributions and biochemical effects of anti-TNF-α nanoparticle stabilized capsules (NPSCs) in mice. It is found that most of the anticipated biochemical changes occur in sub-organ regions that are separate from where the nanomaterials accumulate. In particular, TNF-α-specific lipid biomarker levels change in immune cell-rich regions of organs, while the NPSCs accumulate in spatially isolated filtration regions. Biochemical changes that are associated with the nanomaterials themselves are also observed, demonstrating the power of matrix-assisted laser desorption/ionization (MALDI) MSI to reveal markers indicating possible off-target effects of the delivery agent. This comprehensive assessment using MSI provides spatial context of nanomaterial distributions and efficacy that cannot be easily achieved with other imaging methods, demonstrating the power of MSI to evaluate both expected and unexpected outcomes associated with complex therapeutic delivery systems.

High-content and high-throughput identification of macrophage polarization phenotypes.

Macrophages are plastic cells of the innate immune system that perform a wide range of immune- and homeostasis-related functions. Due to their plasticity, macrophages can polarize into a spectrum of activated phenotypes. Rapid identification of macrophage polarization states provides valuable information for drug discovery, toxicological screening, and immunotherapy evaluation. The complexity associated with macrophage activation limits the ability of current biomarker-based methods to rapidly identify unique activation states. In this study, we demonstrate the ability of a 2-element sensor array that provides an information-rich 5-channel output to successfully determine macrophage polarization phenotypes in a matter of minutes. The simple and robust sensor generates a high dimensional data array which enables accurate macrophage evaluations in standard cell lines and primary cells after cytokine treatment, as well as following exposure to a model disease environment.

Dual Mass Spectrometric Tissue Imaging of Nanocarrier Distributions and Their Biochemical Effects.

Nanomaterial-based drug delivery vehicles are able to deliver therapeutics in a controlled, targeted manner. Currently, however, there are limited analytical methods that can detect both nanomaterial distributions and their biochemical effects concurrently. In this study, we demonstrate that matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and laser ablation inductively coupled plasma mass spectrometry imaging (LA-ICP-MSI) can be used together to obtain nanomaterial distributions and biochemical consequences. These studies employ nanoparticle-stabilized capsules (NPSCs) loaded with siRNA as a testbed. MALDI-MSI experiments on spleen tissues from intravenously injected mice indicate that NPSCs loaded with anti-TNF-α siRNA cause changes to the lipid composition in white pulp regions of the spleen, as anticipated, based on pathways known to be affected by TNF-α, whereas NPSCs loaded with scrambled siRNA do not cause the predicted changes. Interestingly, LA-ICP-MSI experiments reveal that the NPSCs primarily localize in the red pulp, suggesting that the observed changes in lipid composition are due to diffusive rather than localized effects on TNF-α production. Such information is only accessible by combining data from the two modalities, which we accomplish by using the heme signals from MALDI-MSI and iron signals from LA-ICP-MSI to overlay the images. Several unexpected changes in lipid composition also occur in regions where the NPSCs are found, suggesting that the NPSCs themselves can influence tissue biochemistry as well.

Macrophage activation by a substituted pyrimido[5,4-b]indole increases anti-cancer activity.

Immunotherapy has become a promising new approach for cancer treatment due to the immune system's ability to remove tumors in a safe and specific manner. Many tumors express anti-inflammatory factors that deactivate the local immune response or recruit peripheral macrophages into pro-tumor roles. Because of this, effective and specific ways of activating macrophages into anti-tumor phenotypes is highly desirable for immunotherapy purposes. Here, the use of a small molecule TLR agonist as a macrophage activator for anti-cancer therapy is reported. This compound, referred to as PBI1, demonstrated unique activation characteristics and expression patterns compared to treatment with LPS, through activation of TLR4. Furthermore, PBI1 treatment resulted in anti-tumor immune behavior, enhancing macrophage phagocytic efficiency five-fold versus non-treated macrophages. Additive effects were observed via use of a complementary strategy (anti-CD47 antibody), resulting in ∼10-fold enhancement of phagocytosis, suggesting this small molecule approach could be used in conjunction with other therapeutics.

In Vivo Editing of Macrophages through Systemic Delivery of CRISPR-Cas9-Ribonucleoprotein-Nanoparticle Nanoassemblies.

Macrophages are key effectors of host defense and metabolism, making them promising targets for transient genetic therapy. Gene editing through delivery of the Cas9-ribonucleoprotein (RNP) provides multiple advantages over gene delivery-based strategies for introducing CRISPR machinery to the cell. There are, however, significant physiological, cellular, and intracellular barriers to the effective delivery of the Cas9 protein and guide RNA (sgRNA) that have to date, restricted in vivo Cas9 protein-based approaches to local/topical delivery applications. Herein we describe a new nanoassembled platform featuring co-engineered nanoparticles and Cas9 protein that has been developed to provide efficient Cas9-sgRNA delivery and concomitant CRISPR editing through systemic tail-vein injection into mice, achieving >8% gene editing efficiency in macrophages of the liver and spleen.

Nanocapsule-mediated cytosolic siRNA delivery for anti-inflammatory treatment.

The use of nanoparticle-stabilized nanocapsules for cytosolic siRNA delivery for immunomodulation in vitro and in vivo is reported. These NPSCs deliver siRNA directly to the cytosol of macrophages in vitro with concomitant knockdown of gene expression. In vivo studies showed directed delivery of NPSCs to the spleen, enabling gene silencing of macrophages, with preliminary studies showing 70% gene knockdown at a siRNA dose of 0.28 mg/kg. Significantly, the delivery of siRNA targeting tumor necrosis factor-α efficiently silenced TNF-α expression in LPS-challenged mice, demonstrating efficacy in modulating immune response in an organ-selective manner. This research highlights the potential of the NPSC platform for targeted immunotherapy and further manipulation of the immune system.

Surface-Modified Macrophages Facilitate Tracking of Breast Cancer-Immune Interactions.

The immune system has been found to play key roles in cancer development and progression. Macrophages are typically considered to be pro-inflammatory cells but can also facilitate pro-oncogenic activities via associations with tumors and metastases. The study of macrophages and their interactions within the context of cancer microenvironments is stymied by the lack of a system to track them. We present a cell-based strategy for studying cancer-immune cell interactions by chemically modifying the surfaces of macrophages with fluorophores. Two widely used methods are employed, affecting cell surface proteins and glycans via NHS-ester and Staudinger ligation reactions, respectively. We show that these modifications do not interfere with macrophage responses to chemoattractants and that interactions with cancer cells can be readily monitored. This work describes the development of macrophage-based imaging agents for tumor detection and assessment of interactions between immune cells and cancers.

Protein delivery into cells using inorganic nanoparticle-protein supramolecular assemblies.

The delivery of proteins into cells is a potential game changer for a wide array of therapeutic purposes, including cancer therapy, immunomodulation and treatment of inherited diseases. In this review, we present recently developed nanoassemblies for protein delivery that utilize strategies that range from direct assembly, encapsulation and composite formation. We will discuss factors that affect the efficacy of nanoassemblies for delivery from the perspective of both nanoparticles and proteins. Challenges in the field, particularly achieving effective cytosolar protein delivery through endosomal escape or evasion are discussed.

Harnessing Biology to Deliver Therapeutic and Imaging Entities via Cell-Based Methods.

The accumulation of therapeutic and imaging agents at sites of interest is critical to their efficacy. Similarly, off-target effects (especially toxicity) are a major liability for these entities. For this reason, the use of delivery vehicles to improve the distribution characteristics of bio-active agents has become ubiquitous in the field. However, the majority of traditionally employed, cargo-bearing platforms rely on passive accumulation. Even in cases where "targeting" functionalities are used, the agents must first reach the site in order for the ligand-receptor interaction to occur. The next stage of vehicle development is the use of "recruited" entities, which respond to biological signals produced in the tissues to be targeted, resulting in improved specificities. Recently, many advances have been made in the utilization of cells as delivery agents. They are biocompatible, exhibit excellent circulation lifetimes and tissue penetration capabilities, and respond to chemotactic signals. In this Minireview, we will explore various cell types, modifications, and applications where cell-based delivery agents are used.

CRISPRed Macrophages for Cell-Based Cancer Immunotherapy.

We present here an integrated nanotechnology/biology strategy for cancer immunotherapy that uses arginine nanoparticles (ArgNPs) to deliver CRISPR-Cas9 gene editing machinery into cells to generate SIRP-α knockout macrophages. The NP system efficiently codelivers single guide RNA (sgRNA) and Cas9 protein required for editing to knock out the "don't eat me signal" in macrophages that prevents phagocytosis of cancer cells. Turning off this signal increased the innate phagocytic capabilities of the macrophages by 4-fold. This improved attack and elimination of cancer cells makes this strategy promising for the creation of "weaponized" macrophages for cancer immunotherapy.

Effects of engineered nanoparticles on the innate immune system.

Engineered nanoparticles (NPs) have broad applications in industry and nanomedicine. When NPs enter the body, interactions with the immune system are unavoidable. The innate immune system, a non-specific first line of defense against potential threats to the host, immediately interacts with introduced NPs and generates complicated immune responses. Depending on their physicochemical properties, NPs can interact with cells and proteins to stimulate or suppress the innate immune response, and similarly activate or avoid the complement system. NPs size, shape, hydrophobicity and surface modification are the main factors that influence the interactions between NPs and the innate immune system. In this review, we will focus on recent reports about the relationship between the physicochemical properties of NPs and their innate immune response, and their applications in immunotherapy.

Simultaneous cytosolic delivery of a chemotherapeutic and siRNA using nanoparticle-stabilized nanocapsules.

We report on nanoparticle-stabilized capsules (NPSCs) as a platform for the co-delivery of survivin-targeted siRNA and tamoxifen. These capsules feature an inner oil core that provides a carrier for tamoxifen, and is coated on the surface with positively charged nanoparticles self-assembled with siRNA. The multifaceted chemical nature of the NPSC system enables the simultaneous delivery of both payloads directly into the cytosol in vitro. The NPSC co-delivery of tamoxifen and survivin-targeted siRNA into breast cancer cells disables the pathways that inhibit apoptosis, resulting in enhanced breast cell death.

Chemically Engineered Nanoparticle-Protein Interface for Real-Time Cellular Oxidative Stress Monitoring.

A co-engineered nanoparticle/protein peroxide detector is created. This system features a gold nanoparticle functionalized with a galactose headgroup (AuNP-Gal) that reacts covalently with a boronate-modified green fluorescent protein (PB-GFP). Boronate acid-saccharide complexation between PB-GFP and AuNP-Gal affords a highly stable assembly. This complex is disrupted by peroxide, allowing quantitative and selective monitoring of hydrogen peroxide production in real time.

Progress and perspective of inorganic nanoparticle-based siRNA delivery systems.

INTRODUCTION: Small interfering RNA (siRNA) is an effective method for regulating the expression of proteins, even "undruggable" ones that are nearly impossible to target through traditional small molecule therapeutics. Delivery to the cell and then to the cytosol is the primary requirement for realization of therapeutic potential of siRNA. AREAS COVERED: We summarize recent advances in the design of inorganic nanoparticle with surface functionality and physicochemical properties engineered for siRNA delivery. Specifically, we discuss the main approaches developed so far to load siRNA into/onto NPs, and NP surface chemistry engineered for enhanced intracellular siRNA delivery, endosomal escape, and targeted delivery of siRNA to disease cells and tissues. EXPERT OPINION: Several challenges remain in developing inorganic NPs for efficient and effective siRNA delivery. Getting the material to the chosen site is important, however the greatest hurdle may well be delivery into the cytosol, either through efficient endosomal escape or by direct cytosolic siRNA delivery. Effective delivery at the organismic and cellular level coupled with biocompatible vehicles with low immunogenic response will facilitate the clinical translation of RNAi for the treatment of genetic diseases.