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Palmar-plantar erythrodysesthesia (PPE), also known as hand and foot syndrome, is a condition characterized by inflammation-mediated damage to the skin on the palms and soles of the hands and feet. PPE limits the successful therapeutic applications of anticancer drugs. However, identifying this toxicity during preclinical studies is challenging due to the lack of accurate in vitro and in vivo animal-based models. Therefore, there is a need for reliable models that would allow the detection of this toxicity early during the drug development process. Herein, we describe the use of an in vitro skin explant assay to assess traditional DXR, Doxil reference listed drug (RLD) and two generic PEGylated liposomal DXR formulations for their abilities to cause inflammation and skin damage. We demonstrate that the results obtained with the in vitro skin explant assay model for traditional DXR and Doxil correlate with the clinical data., Competing Interests: Declaration of competing interest A.I., M.F., S.Sh., and A.D. are employees of Alcyomics Ltd., a contract research organization that owns the intellectual property and receives royalties from using the skin explant assay (trading as Skimune®.) All other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Immunostimulation caused by nanoparticles may be beneficial or adverse depending on their intended application. Activation of immune cells is beneficial for indications targeting the immune system for therapeutic purposes, such as tumor microenvironment reprogramming, immunotherapy, and vaccines. When it is unwanted, however, immunostimulation may lead to excessive inflammation, cytokine storm, and hypersensitivity reactions. The increasing use of silica nanoparticles (SiNPs) for the delivery of drugs, imaging agents, and antigens warrants preclinical studies aimed at understanding carrier-mediated effects on the number, activation status, and function of immune cell subsets. Herein, we present an in vitro study utilizing primary human peripheral blood mononuclear cells (PBMC) to investigate the proinflammatory properties of four types of SiNPs varying in size and porosity. Cytokine analysis was performed in resting and LPS-primed PBMC cultures to understand the ability of silica nanoparticles to induce de novo and exaggerate preexisting inflammation, respectively. Changes in the number and activation status of lymphoid and myeloid cells were studied by flow cytometry to gain further insight into SiNP-mediated immunostimulation. Nonporous SiNPs were found to be more proinflammatory than mesoporous SiNPs, and larger-sized particles induced greater cytokine response. LPS-primed PBMC resulted in increased susceptibility to SiNPs. Immunophenotyping analysis of SiNP-treated PBMC resulted in T and B lymphocyte, natural killer cell, and dendritic cell activation. Additionally, a loss of regulatory T cells and an increase in γδ TCR T cell population were observed with all particles. These findings have implications for the utility of SiNPs for the delivery of drugs and imaging agents.

Cowpea mosaic virus (CPMV) is a plant virus that is currently being developed for intratumoral immunotherapy. CPMV relieves the immune system from tumor-induced immunosuppression; reprograms the tumor microenvironment to an activated state whereby the treated and distant tumors are recognized and eradicated. Toward translational studies, we investigated the safety of CPMV, specifically addressing whether pathogenicity would be induced in mammalian cells. We show that murine macrophage immune cells recognize CPMV; however, there is no indication of de novo viral protein synthesis or RNA replication. Furthermore, we show that CPMV does not induce hemolysis, platelet aggregation and plasma coagulation amongst other assays in human blood and immune cells. Taken together, we anticipate that these results will reinforce the development of CPMV as an immunotherapeutic platform., (© 2024. The Author(s).)

Silica nanoparticles are increasingly considered for drug delivery applications. These applications require an understanding of their biocompatibility, including their interactions with the immune system. However, systematic studies for silica nanoparticle immunological safety profiles are lacking. To fill this gap, we conducted an in vitro study investigating various aspects of silica nanoparticles' interactions with blood and immune cells. Four types of silica nanoparticles with variations in size and porosity were studied. These included nonporous Stöber silica nanoparticles with average diameters of approximately 50 and 100 nm (SNP50 and SNP100), mesoporous silica nanoparticles of approximately 100 nm (Meso100), and hollow mesoporous silica nanoparticles of approximately 100 nm (HMSNP100) in diameter, respectively. The hematological compatibility was assessed using hemolysis, complement activation, platelet aggregation, and plasma coagulation assays. The effects of nanoparticles on immune cell function were studied using in vitro phagocytosis, chemotaxis, natural killer cell cytotoxicity, leukocyte proliferation, human lymphocyte activation, colony-forming unit granulocyte-macrophage, and leukocyte procoagulant activity assays. The in vitro findings suggest that at high concentrations, corresponding to the in vivo human dose of 40 mg/kg, silica nanoparticles demonstrated an array of immunotoxic effects that depended on their physicochemical properties. However, all types of silica nanoparticles studied were not immunotoxic at concentrations corresponding to lower doses (≤ 8 mg/kg) comparable to that of nanocarriers in other nanomedicines currently used in the clinic. These findings are promising for using silica nanoparticles for the systemic delivery of bioactive and imaging agents.

Nano-mupirocin is a PEGylated nano-liposomal formulation of the antibiotic mupirocin, undergoing evaluation for treating infectious diseases and intratumor bacteria. Intratumoral microbiota play an important role in the regulation of tumor progression and therapeutic efficacy. However, antibiotic use to target intratumoral bacteria should be performed in a way that will not affect the gut microbiota, found to enable the efficacy of cancer treatments. Nano-mupirocin may offer such a selective treatment. Herein, we demonstrate the ability of Nano-mupirocin to successfully target tumor-residing Fusobacterium nucleatum without an immediate effect on the gut microbiome. In-depth characterization of this novel formulation was performed, and the main findings include: (i). the pharmacokinetic analysis of mupirocin administered as Nano-mupirocin vs mupirocin lithium (free drug) demonstrated that most of the Nano-mupirocin in plasma is liposome associated; (ii). microbiome analysis of rat feces showed no significant short-term difference between Nano-mupirocin, mupirocin lithium and controls; (iii). Nano-mupirocin was active against intratumoral F. nucleatum, a tumor promoting bacteria that accumulates in tumors of the AT3 mice model of breast cancer. These data suggest the ability of Nano-mupirocin to target tumor residing and promoting bacteria., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cowpea mosaic virus (CPMV) has demonstrated superior immune stimulation and efficacy as an intratumoral immunotherapy, providing a strong argument for its clinical translation. One important consideration for any new drug candidate is the long-term stability of the drug and its formulation. Therefore, our lab has evaluated the physical stability and biological activity, that is, anti-tumor potency, of formulations of CPMV in buffer (with and without a sucrose preservative) in multiple temperature conditions ranging from ultralow freezers to a heated incubator over a period of 9 months. We found that non-refrigerated temperatures 37°C and room temperature quickly led to CPMV destabilization, as evidenced by significant protein and RNA degradation after just 1 week. Refrigerated storage at 4°C extended physical stability, though signs of particle breakage and RNA escape appeared after 6 and 9 months. CPMV stored in frozen conditions, including -20°C, -80°C, and liquid N 2 , remained intact and matched the characteristics of fresh CPMV throughout the duration of the study. The biological activity was evaluated using a murine dermal melanoma model, and efficacy followed the observed trends in physical stability: CPMV stored in refrigerated and warmer conditions exhibited decreased anti-tumor efficacy compared to freshly prepared formulations. Meanwhile, frozen-stored CPMV performed similarly to freshly purified CPMV, resulting in reduced tumor growth and extended survival. Data, therefore, indicates that CPMV stored long-term in cold or frozen conditions remains stable and efficacious, providing additional support to advance this powerful plant virus to translation., Competing Interests: The authors declare the following competing financial interest(s): Dr. Nicole F. Steinmetz is a co‐founder of, has equity in, and has a financial interest with Mosaic ImmunoEngineering Inc. Dr. Nicole F. Steinmetz is a co‐founder of, and serves as manager of Pokometz Scientific LLC, under which she is a paid consultant to Mosaic ImmunoEngineering Inc., Flagship Labs 95 Inc., and Arana Biosciences Inc. The other authors declare no potential conflict of interest., (© 2024 The Author(s). Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of The American Institute of Chemical Engineers.)

Fibrous nanomaterials containing silica, titanium oxide, and carbon nanotubes are notoriously known for their undesirable inflammatory responses and associated toxicities that have been extensively studied in the environmental and occupational toxicology fields. Biopersistance and inflammation of "hard" nanofibers prevent their broader biomedical applications. To utilize the structural benefits of fibrous nanomaterials for functionalization with moieties of therapeutic significance while preventing undesirable immune responses, researchers employ natural biopolymers─RNA and DNA─to design "soft" and biodegradable nanomaterials with controlled immunorecognition. Nucleic acid nanofibers have been shown to be safe and efficacious in applications that do not require their delivery into the cells such as the regulation of blood coagulation. Previous studies demonstrated that unlike traditional therapeutic nucleic acids (e.g., CpG DNA oligonucleotides) nucleic acid nanoparticles (NANPs), when used without a carrier, are not internalized by the immune cells and, as such, do not induce undesirable cytokine responses. In contrast, intracellular delivery of NANPs results in cytokine responses that are dependent on the physicochemical properties of these nanomaterials. However, the structure-activity relationship of innate immune responses to intracellularly delivered fibrous NANPs is poorly understood. Herein, we employ the intracellular delivery of model RNA/DNA nanofibers functionalized with G-quadruplex-based DNA aptamers to investigate how their structural properties influence cytokine responses. We demonstrate that nanofibers' scaffolds delivered to the immune cells using lipofectamine induce interferon response via the cGAS-STING signaling pathway activation and that DNA aptamers incorporation shields the fibers from recognition by cGAS and results in a lower interferon response. This structure-activity relationship study expands the current knowledge base to inform future practical applications of intracellularly delivered NANPs as vaccine adjuvants and immunotherapies.

The use of nanoparticles as drug delivery carriers requires analysis of their safety, which among other tests, includes immunotoxicity. Nanoparticles are also increasingly used for applications intended to specifically activate, inhibit, or modify the immune system's responses to improve the treatment of inflammatory and autoimmune disorders, cancer immunotherapy, and vaccines targeting cancer cells and viral and bacterial pathogens. In addition to the safety, the analysis of nanoparticles intended for immune system targeting includes mechanistic immunology investigations. Immunophenotyping provides researchers with a tool to assess the immune cell viability and activation status. These results provide mechanistic insights into nanoparticle efficacy and toxicity and therefore are of interest to the biomedical nanotechnology field. However, no standardized approaches exist due to the breadth of methods and instruments available for this analysis. This chapter provides detailed instructions for applying this methodology to analyze nanoparticle effects on subsets of immune cells present in peripheral blood. While this experimental strategy is specific to the NovoCyte 3005 flow cytometer, it can be adapted to other instruments. Instructions for instrument setup, calibration, and antibody qualification are described in this book's Chapter 24 , Immunophenotyping, part I., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Monitoring endotoxin contamination in drugs and medical devices is required to avoid pyrogenic responses and septic shock in patients receiving these products. Endotoxin contamination of engineered nanomaterials and nanotechnology-based medical products represents a significant translational hurdle. Nanoparticles often interfere with an in vitro limulus amebocyte lysate (LAL) assay commonly used in the pharmaceutical industry for the detection and quantification of endotoxin. Such interference challenges the preclinical development of nanotechnology-formulated drugs and medical devices containing engineered nanomaterials. Protocols for the analysis of nanoparticles using LAL assays have been reported before. Here, we discuss considerations for selecting an LAL format and describe a few experimental approaches for overcoming nanoparticle interference with the LAL assays to obtain more accurate estimations of endotoxin contamination in nanotechnology-based products. The discussed approaches do not solve all types of nanoparticle interference with the LAL assays but could be used as a starting point to address the problem. This chapter also describes approaches to prevent endotoxin contamination in nanotechnology-formulated products., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Autoimmune responses are characterized by the presence of antibodies and lymphocytes specific to self or so-called autoantigens. Among such autoantigens is DNA; therefore, screening for antibodies recognizing single- and/or double-stranded DNA is commonly used to detect and classify autoimmune diseases. While autoimmunity affects both sexes, females are generally more affected than males, which is recapitulated in some animal models. A variety of factors, including genetic predisposition and the environment, contribute to the development of autoimmune disorders. Since certain drug products may also contribute to the development of autoimmunity, understanding a drug's potential to trigger an autoimmune response is of interest to immunotoxicology. However, models to study autoimmunity are limited, and it is generally agreed that no model can accurately predict autoimmunity in humans. Herein, we present an in vivo protocol utilizing the SJL/J mouse model to study nanoparticles' effects on the development of autoimmune responses. The protocol is adapted from the literature describing the use of this model to study chemically induced lupus., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Polyethylene glycol, or PEG, is common in consumer products, over-the-counter medications, food, and pharmaceutical products. Concerns about PEG immunogenicity and the subsequent negative impact of pre-existing and product-induced antibodies often shadow the benefits of using PEG in nanotechnology-based products. Such anti-PEG antibodies contribute to the accelerated blood clearance of PEGylated nanomedicines and result in premature drug release and antibody-mediated toxicities. Recent data demonstrated that using PEG in COVID-19 lipid nanoparticle-mRNA vaccines is associated with an induction of anti-PEG antibodies in healthy individuals, further contributing to the development or boosting of pre-existing antibodies and increasing the risks of antibody-mediated toxicities to other products containing PEG. Therefore, monitoring the levels of pre-existing and product-induced anti-PEG antibodies provides mechanistic insights for pharmacology, toxicology, and immunological studies of PEGylated drug products., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Psoriasis, an auto-inflammatory disorder, has major manifestations in the skin but can affect other organs. Currently, this condition has no cure, and the treatments include anti-inflammatory medications. Nanoparticles are widely used for drug delivery and have found successful applications in therapy for cancer and infectious diseases. Nanoparticles can also be used to deliver anti-inflammatory drugs to sites of inflammation. Moreover, some nanotechnology platforms possess intrinsic anti-inflammatory properties and may benefit the therapy of inflammation-driven disorders. Herein, we present a protocol to study nanotechnology concepts' anti-inflammatory properties in a chemically-induced psoriasis model., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Nanoparticles are increasingly used in biomedical applications to influence the way the immune system reacts to tumors and infectious disease-causing agents. Nanoparticles not-intended for immunomodulation can also influence immune responses by affecting immune cell subsets' viability and/or activity. While immunophenotyping is commonly used to assess the effects of drugs and nanoparticles on immune cell subsets, no standardized approach exists due to the breadth of available cell models and instrumentation. In this chapter, we describe a protocol for flow cytometer calibration and reagent qualification prior to its use in the immunophenotyping experiment. The strategies described herein can be adapted to other instruments. The subsequent chapter-immunophenotyping part II (Chap. 25 )-provides detailed instructions for applying this methodology to analyze nanoparticle effects on subsets of immune cells present in peripheral blood., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Nanoparticles can be cleared from the circulation and taken up by tissue-resident macrophages. This property can be beneficial when drug or antigen delivery to macrophages is desired; however, rapid clearance of nanoparticles not intended for delivery to immune cells may reduce nanoparticle circulation time and affect the efficacy of nanoparticle-formulated drug products. Therefore, understanding nanoparticles' uptake by macrophages is an essential step in the preclinical development of nanotechnology-based drug products. Understanding the route of nanoparticle uptake by macrophages may also provide mechanistic insights into the immunotoxicity of nanomaterials. The protocol described herein can be used to assess the nanoparticles' uptake by macrophages and understand the involvement of scavenger receptor A1 to inform mechanistic studies., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Oxidative stress is commonly observed in cells following exposure to nanoparticles. Both negative (e.g., cytotoxicity and inflammation) and beneficial (e.g., anti-inflammatory and tumor growth inhibiting) responses have been linked in the literature to oxidative stress, emphasizing the importance of developing methodologies to study this phenomenon in cells following their exposure to nanoparticles. In the protocol described herein, primary human T cells isolated from the peripheral blood of healthy donor volunteers are treated with nanoparticles and controls, and the generation of reactive oxygen species is detected by flow cytometry using CM-H 2 DCFDA reagent., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The complement system is complex and includes two main components: the systemic or plasma complement and the so-called intracellular complement or complosome. The complement proteins expressed by the liver and secreted into blood plasma compose the plasma complement system, whereas complement proteins expressed by and functioning inside the cell represent the intracellular complement. The complement system plays an essential role in host defense; however, complement activation may lead to pathologies when uncontrolled. When such undesirable activation of the plasma complement occurs in response to a drug product, it leads to immediate-type hypersensitivity reactions independent of immunoglobulin E. These reactions are often called complement activation-related pseudoallergy (CARPA). In addition to the blood plasma, the complement protein C3 is found in many cells, including lymphocytes, monocytes, endothelial, and even cancer cells. The activation of the intracellular complement generates split products, which are exported from the cell onto the membrane. Since the activation of the intracellular complement in T lymphocytes was found to correlate with autoimmune disorders, and growing evidence is available for the involvement of T lymphocytes in the development of drug-induced hypersensitivity reactions, understanding the ability of nanomaterials to activate intracellular complement may aid in establishing a long-term safety profile for these materials. This chapter describes a flow cytometry-based protocol for detecting intracellular complement activation by engineered nanomaterials., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Adverse drug effects on immune system function represent a significant concern in the pharmaceutical industry, because 10-20% of drug withdrawal from the market is attributed to immunotoxicity. Immunosuppression is one such adverse effect. The traditional immune function test used to estimate materials' immunosuppression is T cell dependent antibody response (TDAR). This method involves a 28-day in vivo study evaluating the animal's antibody titer to a known antigen (Keyhole Limpet Hemocyanin; KLH) with and without challenge. Due to the limited quantities of novel drug candidates, an in vitro method called human lymphocyte activation (HuLA) assay has been developed to substitute the traditional TDAR assay during early preclinical development. In this test, leukocytes isolated from healthy donors vaccinated with the current year's flu vaccine are incubated with Fluzone in the presence or absence of nanoparticles. The antigen-specific lymphocyte proliferation is then measured by ELISA analyzing incorporation of BrdU into DNA of the proliferating cells. Here we describe the experimental procedures for investigating immunosuppressive properties of nanoparticles by both TDAR and HuLA assays, discuss the in vitro-in vivo correlation of these methods, and show a case study using the iron oxide nanoparticle formulation, Feraheme., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Beta-glucans with diverse chemical structures are produced by a variety of microorganisms and are commonly found in microbial cell walls. β-(1,3)-D-glucans are present in yeast and fungi, and, for this reason, their traces are commonly used as a sign of yeast or fungal infection or contamination. Despite being less immunologically active than endotoxins, beta-glucans are pro-inflammatory and can activate cytokines and other immunological responses via their cognate pattern recognition receptors. Unlike endotoxins, there is no established threshold pyrogen dose for beta-glucans; as such, their quantity in pharmaceutical products is not regulated. Nevertheless, regulatory agencies recognize the potential contribution of beta-glucans to the immunogenicity of protein-containing drug products and recommend assessing beta-glucans to aid the interpretation of immunotoxicity studies and assess the risk of immunogenicity. The protocol for the detection and quantification of β-(1,3)-D-glucans in nanoparticle formulations is based on a modified limulus amoebocyte lysate assay. The results of this test are used to inform immunotoxicity studies of nanotechnology-based drug products., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Delayed-type hypersensitivity (DTH) reactions are among the common reasons for drug withdrawal from clinical use during the post-marketing stage. Several in vivo methods have been developed to test DTH responses in animal models. They include the local lymph node assay (LLNA) and local lymph node proliferation assay (LLNP). While LLNA is instrumental in testing topically administered formulations (e.g., creams), the LLNP was proven to be predictive of drug-mediated DTH in response to small molecule pharmaceuticals. Global efforts in reducing the use of research animals lead to the development of in vitro models to predict test-materials' mediated DTH. Two such models include the analysis of surface marker expression in human cell lines THP-1 and U-937. These tests are known as the human cell line activation test (hCLAT) and myeloid U937 skin sensitization test (MUSST or U-SENS), respectively. Here we describe experimental procedures for all these methods, discuss their in vitro-in vivo correlation, and suggest a strategy for applying these tests to analyze engineered nanomaterials and nanotechnology-formulated drug products., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Alterations in mitochondrial membrane potential are associated with the generation of reactive oxygen species and cell death. While eliminating cancer cells is beneficial for cancer therapy, cytotoxicity to healthy cells may limit the therapeutic applications of mitochondria-damaging nanoparticles. Due to the critical role mitochondria play in cell viability and function, it is important to detect such alterations when studying nanomaterials for therapeutic applications. The protocol described herein utilizes JC-1 dye to detect nanoparticle-mediated changes in mitochondrial membrane potential and is intended to support mechanistic immunotoxicology studies., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Nanoparticles are frequently considered in vaccine applications due to their ability to co-deliver multiple antigens and adjuvants to antigen-presenting cells. Some nanoparticles also have intrinsic adjuvant properties that further enhance their ability to stimulate immune cells. The delivery of tumor-specific antigens to antigen-presenting cells (APCs) with subsequent antigenic peptide presentation in the context of class I major histocompatibility complex (MHC-I) molecules represents an essential effort in developing nanotechnology-based cancer vaccines. Experimental models are, therefore, needed to gauge the efficiency of nanotechnology carriers in achieving peptide antigen delivery to APCs and presentation in the context of MHC-I. The assay described herein utilizes a model antigen ovalbumin and model APCs, murine bone marrow-derived dendritic cells. The 25-D1.16 antibody, specific to the ovalbumin (OVA) MHC-I peptide SIINFEKL, recognizes this peptide presented in the context of the murine H2-K b class I MHC molecule, allowing the presentation of this antigen on APCs to be detected by flow cytometry after nanoparticle delivery., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Nanotechnology for drug delivery has made significant advancements over the last two decades. Innovations have been made in cancer research and development, including chemotherapies, imaging agents, and vaccine strategies, as well as other therapeutic areas, e.g., the recent commercialization of mRNA lipid nanoparticles as vaccines against the SARS-CoV-2 virus. The field has also seen technological advancements to aid in addressing the complex questions posed by these novel therapies. In this latest edition of protocols and methods for nanoparticle characterization, we highlight both old and new methodologies for defining physicochemical properties, present both in vitro and in vivo methods to test for a variety of immunotoxicities, and describe assays used for pharmacological studies to assess drug release and tissue distribution., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The assessment of antigen presentation by dendritic cells and subsequent antigen-dependent activation of T lymphocytes is a critical step underlying the efficacy of nanoparticle-based therapeutic vaccines. Since nanoparticle physicochemical properties determine their interactions with the immune system, the early stages of nanotechnology-based vaccine development commonly involve optimizing the particles' properties to create a formulation with desired stability, antigen release, targeting of desired cell populations, and efficacy. To accelerate this process, in vitro models suitable for the rapid assessment of a novel vaccine candidate's efficacy are highly desirable. One such model is described in this protocol. Herein, nanoparticles are formulated to deliver a model antigen, SIINFEKL (OVA 257-264 ), the immunodominant class I peptide derived from ovalbumin. These nanoparticles are added to the culture of murine bone marrow-derived dendritic cells, which are subsequently co-incubated with CD8 + T cells from OT-I transgenic mice. The efficient antigen presentation by dendritic cells results in the antigen-dependent proliferation of CD8 + T cells, which is detected by flow cytometry., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The induction of oxidative stress by engineered nanomaterials has been associated with cytotoxic and inflammatory responses, damaging healthy cells and tissues. In contrast, when directed against cancer and autoinflammatory diseases, some nanomaterials inducing oxidative stress have also been reported as potential therapies for these disorders. Therefore, studying oxidative stress has become a popular tool not only in toxicology and immunotoxicology but in other areas of biology as well, including those related to developing novel therapies. Total oxidative stress may result from multiple cellular organelles. The protocol described herein allows for the analysis of oxidative stress in mitochondria., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Nanoparticles can be engineered for targeted antigen delivery to immune cells and for stimulating an immune response to improve the antigen immunogenicity. This approach is commonly used to develop nanotechnology-based vaccines. In addition, some nanotechnology platforms may be initially designed for drug delivery, but in the course of subsequent characterization, additional immunomodulatory functions may be discovered that can potentially benefit vaccine efficacy. In both of these scenarios, an in vivo proof of concept study to verify the utility of the nanocarrier for improving vaccine efficacy is needed. Here we describe an experimental approach and considerations for designing an animal study to test adjuvant properties of engineered nanomaterials in vivo., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Immunophenotyping, which is the identification of immune cell subsets based on antigen expression, is an integral technique used to determine changes of cell composition and activation in various disease states or as a response to different stimuli. As nanoparticles are increasingly utilized for diagnostic and therapeutic applications, it is important to develop methodology that allows for the evaluation of their immunological impact. Therefore, the development of techniques such as immunophenotyping are desirable. Currently, the most common technique used to perform immunophenotyping is multicolor flow cytometry., Methods: We developed two distinct multicolor flow cytometry immunophenotyping panels which allow for the evaluation of the effects of nanoparticles on the composition and activation status of treated human peripheral blood mononuclear cells. These two panels assess the presence of various lymphoid and myeloid-derived cell populations as well as aspects of their activation statuses-including proliferation, adhesion, co-stimulation/presentation, and early activation-after treatment with controls or nanoparticles. To conduct assay performance qualification and determine the applicability of this method to preclinical characterization of nanoparticles, we used clinical-grade nanoformulations (AmBisome, Doxil and Feraheme) and research-grade PAMAM dendrimers of different sizes (G3, G4 and G5) and surface functionalities (amine-, carboxy- and hydroxy-)., Results and Discussion: We found that formulations possessing intrinsic fluorescent properties (e.g., Doxil and AmBisome) interfere with accurate immunophenotyping; such interference may be partially overcome by dilution. In the absence of interference (e.g., in the case of dendrimers), nanoparticle size and surface functionalities determine their effects on the cells with large amine-terminated dendrimers being the most reactive., Competing Interests: JZ is employed by Agilent Technologies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Newton, Zhang, Donohue, Unnithan, Cedrone, Xu, Vermilya, Malys, Clogston and Dobrovolskaia.)

This Special Issue is intended to celebrate Professor Yechezkel Barenholz's distinguished achievements [...].

Nucleic acid nanoparticles (NANPs) require a carrier to allow for their intracellular delivery to immune cells. Cytokine production, specifically type I and III interferons, allows for reliable monitoring of the carrier effect on NANP immunostimulation. Recent studies have shown that changes in the delivery platform (e.g., lipid-based carriers vs. dendrimers) can alter NANPs' immunorecognition and downstream cytokine production in various immune cell populations. Herein, we used flow cytometry and measured cytokine induction to show how compositional variations in commercially available lipofectamine carriers impact the immunostimulatory properties of NANPs with different architectural characteristics.

Wireframe DNA origami can be used to fabricate virus-like particles for a range of biomedical applications, including the delivery of nucleic acid therapeutics. However, the acute toxicity and biodistribution of these wireframe nucleic acid nanoparticles (NANPs) have not been previously characterized in animal models. In the present study, we observed no indications of toxicity in BALB/c mice following a therapeutically relevant dosage of nonmodified DNA-based NANPs via intravenous administration, based on liver and kidney histology, liver and kidney biochemistry, and body weight. Further, the immunotoxicity of these NANPs was minimal, as indicated by blood cell counts and type-I interferon and pro-inflammatory cytokines. In an SJL/J model of autoimmunity, we observed no indications of NANP-mediated DNA-specific antibody response or immune-mediated kidney pathology following the intraperitoneal administration of NANPs. Finally, biodistribution studies revealed that these NANPs accumulate in the liver within one hour, concomitant with substantial renal clearance. Our observations support the continued development of wireframe DNA-based NANPs as next-generation nucleic acid therapeutic delivery platforms.

Cancer-targeted therapy by a chemotherapeutic agent formulated in a nanoscale platform has been challenged by complex and inefficient manufacturing, low drug loading, difficult characterization, and marginally improved therapeutic efficacy. This study investigated facile-to-produce nanocomplexes of doxorubicin (DOX), a widely used cancer drug, and clinically approved DNA fragments that are extracted from a natural source. DOX was found to self-assemble DNA fragments into relatively monodispersed nanocomplexes with a diameter of ∼70 nm at 14.3% (w/w) drug loading by simple and scalable mixing. The resulting DOX/DNA nanocomplexes showed sustained DOX release, unlike overly stable Doxil®, cellular uptake via multiple endocytosis pathways, and high hematological and immunological compatibility. DOX/DNA nanocomplexes eradicated EL4 T lymphoma cells in a time-dependent manner, eventually surpassing free DOX. Extended circulation of DOX/DNA nanocomplexes, while avoiding off-target accumulation in the lung and being cleared from the liver, resulted in rapid accumulation in tumor and lowered cardio toxicity. Finally, tumor growth of EL4-challenged C57BL/6 mice (syngeneic model) and OPM2-challenged NSG mice (human xenograft model) were efficiently inhibited by DOX/DNA nanocomplexes with enhanced overall survival, in comparison with free DOX and Doxil®, especially upon repeated administrations. DOX/DNA nanocomplexes are a promising chemotherapeutics delivery platform for their ease of manufacturing, high biocompatibility, desired drug release and accumulation, efficient tumor eradication with improved safety, and further engineering versatility for extended therapeutic applications., Competing Interests: Declaration of Competing Interest The authors declare the following competing financial interest(s): J.K., S.B., and I.S.K. are employees of Pharma Research, Co., Ltd., a company developing DNA-based medical devices, cosmetics, and pharmaceuticals. Y.J.K. is a consultant for Pharma Research, Co., Ltd. The potential conflict of interest (COI) aspects of the reported work were disclosed and COI management plans were reviewed and approved by the COI oversight committee at UC Irvine., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Nanoparticles can be used to formulate Toll-like receptor (TLR) agonists as vaccine and immunotherapy adjuvants or contain undesirable contaminants (e.g., endotoxin, CpG DNA, flagellin) with TLR-agonist activity. In both scenarios, the activation of the innate immune pattern recognition receptor leads to the inflammatory response that can be beneficial as in the case with vaccines and immunotherapies or adverse as in the case with contaminants. The protocol described herein utilizes commercially available reporter cell lines expressing individual TLRs, which, upon activation with their cognate agonists, stimulate the cells to produce secreted alkaline phosphatase detectable using a plate reader., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Three-dimensional wireframe DNA origami have programmable structural and sequence features that render them potentially suitable for prophylactic and therapeutic applications. However, their innate immunological properties, which stem from parameters including geometric shape and cytosine-phosphate-guanine dinucleotide (CpG) content, remain largely unknown. Here, we investigate the immunostimulatory properties of 3D wireframe DNA origami on the TLR9 pathway using both reporter cell lines and primary immune cells. Our results suggest that bare 3D polyhedral wireframe DNA origami induce minimal TLR9 activation despite the presence of numerous internal CpG dinucleotides. However, when displaying multivalent CpG-containing ssDNA oligos, wireframe DNA origami induce robust TLR9 pathway activation, along with enhancement of downstream immune response as evidenced by increases in Type I and Type III interferon (IFN) production in peripheral blood mononuclear cells. Further, we find that CpG copy number and spatial organization each contribute to the magnitude of TLR9 signaling and that NANP-attached CpGs do not require phosphorothioate stabilization to elicit signaling. These results suggest key design parameters for wireframe DNA origami that can be programmed to modulate immune pathway activation controllably for prophylactic and therapeutic applications.

Cancer nanotechnologies possess immense potential as therapeutic and diagnostic treatment modalities and have undergone significant and rapid advancement in recent years. With this emergence, the complexities of data standards in the field are on the rise. Data sharing and reanalysis is essential to more fully utilize this complex, interdisciplinary information to answer research questions, promote the technologies, optimize use of funding, and maximize the return on scientific investments. In order to support this, various data-sharing portals and repositories have been developed which not only provide searchable nanomaterial characterization data, but also provide access to standardized protocols for synthesis and characterization of nanomaterials as well as cutting-edge publications. The National Cancer Institute's (NCI) caNanoLab is a dedicated repository for all aspects pertaining to cancer-related nanotechnology data. The searchable database provides a unique opportunity for data mining and the use of artificial intelligence and machine learning, which aims to be an essential arm of future research studies, potentially speeding the design and optimization of next-generation therapies. It also provides an opportunity to track the latest trends and patterns in nanomedicine research. This manuscript provides the first look at such trends extracted from caNanoLab and compares these to similar metrics from the NCI's Nanotechnology Characterization Laboratory, a laboratory providing preclinical characterization of cancer nanotechnologies to researchers around the globe. Together, these analyses provide insight into the emerging interests of the research community and rise of promising nanoparticle technologies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Nucleic acid nanoparticles, or NANPs, rationally designed to communicate with the human immune system, can offer innovative therapeutic strategies to overcome the limitations of traditional nucleic acid therapies. Each set of NANPs is unique in their architectural parameters and physicochemical properties, which together with the type of delivery vehicles determine the kind and the magnitude of their immune response. Currently, there are no predictive tools that would reliably guide the design of NANPs to the desired immunological outcome, a step crucial for the success of personalized therapies. Through a systematic approach investigating physicochemical and immunological profiles of a comprehensive panel of various NANPs, the research team developes and experimentally validates a computational model based on the transformer architecture able to predict the immune activities of NANPs. It is anticipated that the freely accessible computational tool that is called an "artificial immune cell," or AI-cell, will aid in addressing the current critical public health challenges related to safety criteria of nucleic acid therapies in a timely manner and promote the development of novel biomedical tools., (© 2022 The Authors. Small published by Wiley-VCH GmbH.)

Different therapeutic nucleic acids (TNAs) can be unified in a single structure by their elongation with short oligonucleotides designed to self-assemble into nucleic acid nanoparticles (NANPs). With this approach, therapeutic cocktails with precisely controlled composition and stoichiometry of active ingredients can be delivered to the same diseased cells for enhancing pharmaceutical action. In this work, an additional nanotechnology-based therapeutic option that enlists a biocompatible NANP-encoded platform for their controlled patient-specific immunorecognition is explored. For this, a set of representative functional NANPs is extensively characterized in vitro, ex vivo, and in vivo and then further analyzed for immunostimulation of human peripheral blood mononuclear cells freshly collected from healthy donor volunteers. The results of the study present the advancement of the current TNA approach toward personalized medicine and offer a new strategy to potentially address top public health challenges related to drug overdose and safety through the biodegradable nature of the functional platform with immunostimulatory regulation., Competing Interests: Conflict of Interest The authors declare no conflict of interest.

Nanotechnology carriers have become common in pharmaceutical products because of their benefits to drug delivery, including reduced toxicities and improved efficacy of active pharmaceutical ingredients due to targeted delivery, prolonged circulation time, and controlled payload release. While available examples of reduced drug toxicity through formulation using a nanocarrier are encouraging, current data also demonstrate that nanoparticles may change a drug's biodistribution and alter its toxicity profile. Moreover, individual components of nanoparticles and excipients commonly used in formulations are often not immunologically inert and contribute to the overall immune responses to nanotechnology-formulated products. Said immune responses may be beneficial or adverse depending on the indication, dose, dose regimen, and route of administration. Therefore, comprehensive toxicology studies are of paramount importance even when previously known drugs, components, and excipients are used in nanoformulations. Recent data also suggest that, despite decades of research directed at hiding nanocarriers from the immune recognition, the immune system's inherent property of clearing particulate materials can be leveraged to improve the therapeutic efficacy of drugs formulated using nanoparticles. Herein, I review current knowledge about nanoparticles' interaction with the immune system and how these interactions contribute to nanotechnology-formulated drug products' safety and efficacy through the lens of over a decade of nanoparticle characterization at the Nanotechnology Characterization Laboratory., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dobrovolskaia.)

The unbalanced coagulation of blood is a life-threatening event that requires accurate and timely treatment. We introduce a user-friendly biomolecular platform based on modular RNA-DNA anticoagulant fibers programmed for reversible extracellular communication with thrombin and subsequent control of anticoagulation via a "kill-switch" mechanism that restores hemostasis. To demonstrate the potential of this reconfigurable technology, we designed and tested a set of anticoagulant fibers that carry different thrombin-binding aptamers. All fibers are immunoquiescent, as confirmed in freshly collected human peripheral blood mononuclear cells. To assess interindividual variability, the anticoagulation is confirmed in the blood of human donors from the U.S. and Brazil. The anticoagulant fibers reveal superior anticoagulant activity and prolonged renal clearance in vivo in comparison to free aptamers. Finally, we confirm the efficacy of the "kill-switch" mechanism in vivo in murine and porcine models.

Pharmaceutical products can activate immune cells, suppress their function, or change the immune responses to traditional immunologically active agonists such as those present in microbes. Therefore, the assessment of immunostimulation, immunosuppression, and immunomodulation comprises the backbone of immunotoxicity studies of new drug entities. Depending on physicochemical properties (e.g., size, charge, surface functionalities, hydrophobicity), nanoparticles can be immunostimulatory, immunosuppressive, and immunomodulatory. Various methods and experimental frameworks have been established to support preclinical translational studies of nanotechnology-based drug products. Immunophenotyping after the exposure of cells or preclinical animal models to nanoparticles can provide critical information about the changes in both the numbers of immune cells and their activation status. However, this methodology is underutilized in preclinical studies of engineered nanomaterials. Herein, we review current literature about varieties of instrumentation and methods utilized for immunophenotyping, discuss their advantages and limitations, and propose a roadmap for applying immunophenotyping to support preclinical immunological characterization of nanotechnology-based formulations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

After over a billion of vaccinations with messenger RNA-lipid nanoparticle (mRNA-LNP) based SARS-CoV-2 vaccines, anaphylaxis and other manifestations of hypersensitivity can be considered as very rare adverse events. Although current recommendations include avoiding a second dose in those with first-dose anaphylaxis, the underlying mechanisms are unknown; therefore, the risk of a future reaction cannot be predicted. Given how important new mRNA constructs will be to address the emergence of new viral variants and viruses, there is an urgent need for clinical approaches that would allow a safe repeated immunization of high-risk individuals and for reliable predictive tools of adverse reactions to mRNA vaccines. In many aspects, anaphylaxis symptoms experienced by the affected vaccine recipients resemble those of infusion reactions to nanomedicines. Here we share lessons learned over a decade of nanomedicine research and discuss the current knowledge about several factors that individually or collectively contribute to infusion reactions to nanomedicines. We aim to use this knowledge to inform the SARS-CoV-2 lipid-nanoparticle-based mRNA vaccine field., (© 2022. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Recent advances in nanotechnology now allow for the methodical implementation of therapeutic nucleic acids (TNAs) into modular nucleic acid nanoparticles (NANPs) with tunable physicochemical properties which can match the desired biological effects, provide uniformity, and regulate the delivery of multiple TNAs for combinatorial therapy. Despite the potential of novel NANPs, the maintenance of their structural integrity during storage and shipping remains a vital issue that impedes their broader applications. Cold chain storage is required to maintain the potency of NANPs in the liquid phase, which greatly increases transportation costs. To promote long-term storage and retention of biological activities at higher temperatures (e.g., +50 °C), a panel of representative NANPs is first exposed to three different drying mechanisms-vacuum concentration (SpeedVac), lyophilization (Lyo), and light-assisted drying (LAD)-and then rehydrated and analyzed. While SpeedVac primarily operates using heat, Lyo avoids temperature increases by taking advantage of pressure reduction and LAD involves a near-infrared laser for uniform drying in the presence of trehalose. This work compares and defines refinements crucial in formulating an optimal strategy for producing stable, fully functional NANPs and presents a forward advancement in their development for clinical applications., (© 2022 Wiley-VCH GmbH.)

With numerous recent advances, the field of therapeutic nucleic acid nanotechnology is now poised for clinical translation supported by several examples of FDA-approved nucleic acid nanoformulations including two recent mRNA-based COVID-19 vaccines. Within this rapidly growing field, a new subclass of nucleic acid therapeutics called nucleic acid nanoparticles (NANPs) has emerged in recent years, which offers several unique properties distinguishing it from traditional therapeutic nucleic acids. Key unique aspects of NANPs include their well-defined 3D structure, their tunable multivalent architectures, and their ability to incorporate conditional activations of therapeutic targeting and release functions that enable diagnosis and therapy of cancer, regulation of blood coagulation disorders, as well as the development of novel vaccines, immunotherapies, and gene therapies. However, non-consolidated research developments of this highly interdisciplinary field create crucial barriers that must be overcome in order to impact a broader range of clinical indications. Forming a consortium framework for nucleic acid nanotechnology would prioritize and consolidate translational efforts, offer several unifying solutions to expedite their transition from bench-to-bedside, and potentially decrease the socio-economic burden on patients for a range of conditions. Herein, we review the unique properties of NANPs in the context of therapeutic applications and discuss their associated translational challenges., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Polyethylene glycol or PEG has a long history of use in medicine. Many conventional formulations utilize PEG as either an active ingredient or an excipient. PEG found its use in biotechnology therapeutics as a tool to slow down drug clearance and shield protein therapeutics from undesirable immunogenicity. Nanotechnology field applies PEG to create stealth drug carriers with prolonged circulation time and decreased recognition and clearance by the mononuclear phagocyte system (MPS). Most nanomedicines approved for clinical use and experimental nanotherapeutics contain PEG. Among the most recent successful examples are two mRNA-based COVID-19 vaccines that are delivered by PEGylated lipid nanoparticles. The breadth of PEG use in a wide variety of over the counter (OTC) medications as well as in drug products and vaccines stimulated research which uncovered that PEG is not as immunologically inert as it was initially expected. Herein, we review the current understanding of PEG's immunological properties and discuss them in the context of synthesis, biodistribution, safety, efficacy, and characterization of PEGylated nanomedicines. We also review the current knowledge about immunological compatibility of other polymers that are being actively investigated as PEG alternatives., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Understanding, predicting, and minimizing the immunogenicity of peptide-based therapeutics are of paramount importance for ensuring the safety and efficacy of these products. The so-called anti-drug antibodies (ADA) may have various clinical consequences, including but not limited to the alteration in the product's distribution, biological activity, and clearance profiles. The immunogenicity of biotherapeutics can be influenced by immunostimulation triggered by the presence of innate immune response modulating impurities (IIRMIs) inadvertently introduced during the manufacturing process. Herein, we evaluate the applicability of several in vitro assays (i.e., complement activation, leukocyte proliferation, and cytokine secretion) for the screening of innate immune responses induced by ten common IIRMIs ( Bacillus subtilis flagellin, FSL-1, zymosan, ODN2006, poly(I:C) HMW, poly(I:C) LMW, CLO75, MDP, ODN2216, and Escherichia coli O111:B4 LPS), and a model biotherapeutic Forteo™ (teriparatide). Our study identifies cytokine secretion from healthy human donor peripheral blood mononuclear cells (PBMC) as a sensitive method for the in vitro monitoring of innate immune responses to individual IIRMIs and teriparatide (TP). We identify signature cytokines, evaluate both broad and narrow multiplex cytokine panels, and discuss how the assay logistics influence the performance of this in vitro assay., Competing Interests: The authors declared no conflict of interest.

Innate immunity can be triggered by the presence of microbial antigens and other contaminants inadvertently introduced during the manufacture and purification of bionanopharmaceutical products. Activation of these innate immune responses, including cytokine secretion, complement, and immune cell activation, can result in unexpected and undesirable host immune responses. These innate modulators can also potentially stimulate the activation of adaptive immune responses, including the formation of anti-drug antibodies which can impact drug effectiveness. To prevent induction of these adverse responses, it is important to detect and quantify levels of these innate immunity modulating impurities (IIMIs) that may be present in drug products. However, while it is universally agreed that removal of IIMIs from drug products is crucial for patient safety and to prevent long-term immunogenicity, there is no single assay capable of directly detecting all potential IIMIs or indirectly quantifying downstream biomarkers. Additionally, there is a lack of agreement as to which of the many analytical assays currently employed should be standardized for general IIMI screening. Herein, we review the available literature to highlight cellular and molecular mechanisms underlying IIMI-mediated inflammation and its relevance to the safety and efficacy of pharmaceutical products. We further discuss methodologies used for direct and indirect IIMI identification and quantification.

The relatively straightforward methods of designing and assembling various functional nucleic acids into nanoparticles offer advantages for applications in diverse diagnostic and therapeutic approaches. However, due to the novelty of this approach, nucleic acid nanoparticles (NANPs) are not yet used in the clinic. The immune recognition of NANPs is among the areas of preclinical investigation aimed at enabling the translation of these novel materials into clinical settings. NANPs' interactions with the complement system, coagulation systems, and immune cells are essential components of their preclinical safety portfolio. It has been established that NANPs' physicochemical properties-composition, shape, and size-determine their interactions with immune cells (primarily blood plasmacytoid dendritic cells and monocytes), enable recognition by pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs), and mediate the subsequent cytokine response. However, unlike traditional therapeutic nucleic acids (e.g., CpG oligonucleotides), NANPs do not trigger a cytokine response unless they are delivered into the cells using a carrier. Recently, it was discovered that the type of carrier provides an additional tool for regulating both the spectrum and the magnitude of the cytokine response to NANPs. Herein, we review the current knowledge of NANPs' interactions with various components of the immune system to emphasize the unique properties of these nanomaterials and highlight opportunities for their use in vaccines and immunotherapy.