Locking and Unlocking Thrombin Function Using Immunoquiescent Nucleic Acid Nanoparticles with Regulated Retention In Vivo.

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.

Special Issue "Nanotechnology to Overcome the World's Most Critical Health Issues: Liposomes and Beyond-A Themed Issue Dedicated to Professor Yechezkel Barenholz".

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

Change in Lipofectamine Carrier as a Tool to Fine-Tune Immunostimulation of Nucleic Acid Nanoparticles.

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.

Expanding Structural Space for Immunomodulatory Nucleic Acid Nanoparticles (Nanps) via Spatial Arrangement of Their Therapeutic Moieties.

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.

Artificial Immune Cell, AI-cell, a New Tool to Predict Interferon Production by Peripheral Blood Monocytes in Response to Nucleic Acid Nanoparticles.

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.

Anhydrous Nucleic Acid Nanoparticles for Storage and Handling at Broad Range of Temperatures.

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.

RNA-DNA fibers and polygons with controlled immunorecognition activate RNAi, FRET and transcriptional regulation of NF-κB in human cells.

Nucleic acid-based assemblies that interact with each other and further communicate with the cellular machinery in a controlled manner represent a new class of reconfigurable materials that can overcome limitations of traditional biochemical approaches and improve the potential therapeutic utility of nucleic acids. This notion enables the development of novel biocompatible 'smart' devices and biosensors with precisely controlled physicochemical and biological properties. We extend this novel concept by designing RNA-DNA fibers and polygons that are able to cooperate in different human cell lines and that have defined immunostimulatory properties confirmed by ex vivo experiments. The mutual intracellular interaction of constructs results in the release of a large number of different siRNAs while giving a fluorescent response and activating NF-κB decoy DNA oligonucleotides. This work expands the possibilities of nucleic acid technologies by (i) introducing very simple design principles and assembly protocols; (ii) potentially allowing for a simultaneous release of various siRNAs together with functional DNA sequences and (iii) providing controlled rates of reassociation, stabilities in human blood serum, and immunorecognition.

Innate Immunity Modulating Impurities and the Immunotoxicity of Nanobiotechnology-Based Drug Products.

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 Recognition of and Reactions to Nucleic Acid Nanoparticles by Human Immune Cells.

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.

An In Vitro Assessment of Immunostimulatory Responses to Ten Model Innate Immune Response Modulating Impurities (IIRMIs) and Peptide Drug Product, Teriparatide.

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.

Induction of Cytokines by Nucleic Acid Nanoparticles (NANPs) Depends on the Type of Delivery Carrier.

Recent insights into the immunostimulatory properties of nucleic acid nanoparticles (NANPs) have demonstrated that variations in the shape, size, and composition lead to distinct patterns in their immunostimulatory properties. While most of these studies have used a single lipid-based carrier to allow for NANPs' intracellular delivery, it is now apparent that the platform for delivery, which has historically been a hurdle for therapeutic nucleic acids, is an additional means to tailoring NANP immunorecognition. Here, the use of dendrimers for the delivery of NANPs is compared to the lipid-based platform and the differences in resulting cytokine induction are presented.

Application of a Scavenger Receptor A1-Targeted Polymeric Prodrug Platform for Lymphatic Drug Delivery in HIV.

We have developed a macromolecular prodrug platform based on poly(l-lysine succinylated) (PLS) that targets scavenger receptor A1 (SR-A1), a receptor expressed by myeloid and endothelial cells. We demonstrate the selective uptake of PLS by murine macrophage, RAW 264.7 cells, which was eliminated upon cotreatment with the SR-A inhibitor polyinosinic acid (poly I). Further, we observed no uptake of PLS in an SR-A1-deficient RAW 264.7 cell line, even after 24 h incubation. In mice, PLS distributed to lymphatic organs following i.v. injection, as observed by ex vivo fluorescent imaging, and accumulated in lymph nodes following both i.v. and i.d. administrations, based on immunohistochemical analysis with high-resolution microscopy. As a proof-of-concept, the HIV antiviral emtricitabine (FTC) was conjugated to the polymer's succinyl groups via ester bonds, with a drug loading of 14.2% (wt/wt). The prodrug (PLS-FTC) demonstrated controlled release properties in vitro with a release half-life of 15 h in human plasma and 29 h in esterase-inhibited plasma, indicating that drug release occurs through both enzymatic and nonenzymatic mechanisms. Upon incubation of PLS-FTC with human peripheral blood mononuclear cells (PBMCs), the released drug was converted to the active metabolite FTC triphosphate. In a pharmacokinetic study in rats, the prodrug achieved ∼7-19-fold higher concentrations in lymphatic tissues compared to those in FTC control, supporting lymphatic-targeted drug delivery. We believe that the SR-A1-targeted macromolecular PLS prodrug platform has extraordinary potential for the treatment of infectious diseases.

Detection of Beta-Glucan Contamination in Nanotechnology-Based Formulations.

Understanding the potential contamination of pharmaceutical products with innate immunity modulating impurities (IIMIs) is essential for establishing their safety profiles. IIMIs are a large family of molecules with diverse compositions and structures that contribute to the immune-mediated adverse effects (IMAE) of drug products. Pyrogenicity (the ability to induce fever) and activation of innate immune responses underlying both acute toxicities (e.g., anaphylactoid reactions or pseudoallergy, cytokine storm) and long-term effects (e.g., immunogenicity) are among the IMAE commonly related to IIMI contamination. Endotoxins of gram-negative bacteria are the best-studied IIMIs in that both methodologies for and pitfalls in their detection and quantification are well established. Additionally, regulatory guidance documents and research papers from laboratories worldwide are available on endotoxins. However, less information is currently known about other IIMIs. Herein, we focus on one such IIMI, namely, beta-glucans, and review literature and discuss the experience of the Nanotechnology Characterization Lab (NCL) with the detection of beta-glucans in nanotechnology-based drug products.

PEGylated Liposomal Methyl Prednisolone Succinate does not Induce Infusion Reactions in Patients: A Correlation Between in Vitro Immunological and in Vivo Clinical Studies.

PEGylated nanomedicines are known to induce infusion reactions (IRs) that in some cases can be life-threatening. Herein, we report a case study in which a patient with rare mediastinal and intracardiac IgG4-related sclerosing disease received 8 treatments of intravenously administered PEGylated liposomal methylprednisolone-succinate (NSSL-MPS). Due to the ethical requirements to reduce IRs, the patient received a cocktail of premedication including low dose of steroids, acetaminophen and H2 blockers before each infusion. The treatment was well-tolerated in that IRs, complement activation, anti-PEG antibodies and accelerated blood clearance of the PEGylated drug were not detected. Prior to the clinical study, an in vitro panel of assays utilizing blood of healthy donors was used to determine the potential of a PEGylated drug to activate complement system, elicit pro-inflammatory cytokines, damage erythrocytes and affect various components of the blood coagulation system. The overall findings of the in vitro panel were negative and correlated with the results observed in the clinical phase.

Functionally-interdependent shape-switching nanoparticles with controllable properties.

We introduce a new concept that utilizes cognate nucleic acid nanoparticles which are fully complementary and functionally-interdependent to each other. In the described approach, the physical interaction between sets of designed nanoparticles initiates a rapid isothermal shape change which triggers the activation of multiple functionalities and biological pathways including transcription, energy transfer, functional aptamers and RNA interference. The individual nanoparticles are not active and have controllable kinetics of re-association and fine-tunable chemical and thermodynamic stabilities. Computational algorithms were developed to accurately predict melting temperatures of nanoparticles of various compositions and trace the process of their re-association in silico. Additionally, tunable immunostimulatory properties of described nanoparticles suggest that the particles that do not induce pro-inflammatory cytokines and high levels of interferons can be used as scaffolds to carry therapeutic oligonucleotides, while particles with strong interferon and mild pro-inflammatory cytokine induction may qualify as vaccine adjuvants. The presented concept provides a simple, cost-effective and straightforward model for the development of combinatorial regulation of biological processes in nucleic acid nanotechnology.

Bridging communities in the field of nanomedicine.

An early dialogue between nanomedicine developers and regulatory authorities are of utmost importance to anticipate quality and safety requirements for these innovative health products. In order to stimulate interactions between the various communities involved in a translation of nanomedicines to clinical applications, the European Commission's Joint Research Centre hosted a workshop titled "Bridging communities in the field of Nanomedicine" in Ispra/Italy on the 27th -28th September 2017. Experts from regulatory bodies, research institutions and industry came together to discuss the next generation of nanomedicines and their needs to obtain regulatory approval. The workshop participants came up with recommendations highlighting methodological gaps that should be addressed in ongoing projects addressing the regulatory science of nanomedicines. In addition, individual opinions of experts relevant to progress of the regulatory science in the field of nanomedicine were summarised in the format of a survey.

Genotoxicity of amorphous silica nanoparticles: Status and prospects.

Amorphous silica nanoparticles (SNPs) are widely used in biomedical applications and consumer products. Little is known, however, about their genotoxicity and potential to induce gene expression regulation. Despite recent efforts to study the underlying mechanisms of genotoxicity of SNPs, inconsistent results create a challenge. A variety of factors determine particle-cell interactions and underlying mechanisms. Further, high-throughput studies are required to carefully assess the impact of silica nanoparticle physicochemical properties on induction of genotoxic response in different cell lines and animal models. In this article, we review the strategies available for evaluation of genotoxicity of nanoparticles (NPs), survey current status of silica nanoparticle gene alteration and genotoxicity, discuss particle-mediated inflammation as a contributing factor to genotoxicity, identify existing gaps and suggest future directions for this research.

Nanoparticle physicochemical properties determine the activation of intracellular complement.

The complement system plays an essential role in both innate and adaptive immunity. The traditional understanding of this system comes from studies investigating complement proteins produced by the liver and present in plasma to "complement" the immune cell-mediated response to invading pathogens. Recently, it has been reported that immune cells including, but not limited to, T-cells and monocytes, express complement proteins. This complement is referred to as intracellular (IC) and implicated in the regulation of T-cell activation. The mechanisms and the structure-activity relationship between nanomaterials and IC, however, are currently unknown. Herein, we describe a structure-activity relationship study demonstrating that under in vitro conditions, only polymeric materials with cationic surfaces activate IC in T-cells. The effect also depends on particle size and occurs through a mechanism involving membrane damage, thereby IC on the cell surface serves as a self-opsonization marker in response to the nanoparticle-triggered danger affecting the cell integrity.

Structure and Composition Define Immunorecognition of Nucleic Acid Nanoparticles.

Nucleic acid nanoparticles (NANPs) have evolved as a new class of therapeutics with the potential to detect and treat diseases. Despite tremendous advancements in NANP development, their immunotoxicity, one of the major impediments in clinical translation of traditional therapeutic nucleic acids (TNAs), has never been fully characterized. Here, we describe the first systematically studied immunological recognition of 25 representative RNA and DNA NANPs selected to have different design principles and physicochemical properties. We discover that, unlike traditional TNAs, NANPs used without a delivery carrier are immunoquiescent. We show that interferons (IFNs) are the key cytokines triggered by NANPs after their internalization by phagocytic cells, which agrees with predictions based on the experiences with TNAs. However, in addition to type I IFNs, type III IFNs also serve as reliable biomarkers of NANPs, which is usually not characteristic of TNAs. We show that overall immunostimulation relies on NANP shapes, connectivities, and compositions. We demonstrate that, like with traditional TNAs, plasmacytoid dendritic cells serve as the primary interferon producers among all peripheral blood mononuclear cells treated with NANPs, and scavenger receptor-mediated uptake and endosomal Toll-like receptor signaling are essential for NANP immunorecognition. The TLR involvement, however, is different from that expected for traditional TNA recognition. Based on these results, we suggest that NANP technology may serve as a prototype of auxiliary molecular language for communication with the immune system and the modulation of immune responses.

Nucleic Acid Nanoparticles at a Crossroads of Vaccines and Immunotherapies.

Vaccines and immunotherapies involve a variety of technologies and act through different mechanisms to achieve a common goal, which is to optimize the immune response against an antigen. The antigen could be a molecule expressed on a pathogen (e.g., a disease-causing bacterium, a virus or another microorganism), abnormal or damaged host cells (e.g., cancer cells), environmental agent (e.g., nicotine from a tobacco smoke), or an allergen (e.g., pollen or food protein). Immunogenic vaccines and therapies optimize the immune response to improve the eradication of the pathogen or damaged cells. In contrast, tolerogenic vaccines and therapies retrain or blunt the immune response to antigens, which are recognized by the immune system as harmful to the host. To optimize the immune response to either improve the immunogenicity or induce tolerance, researchers employ different routes of administration, antigen-delivery systems, and adjuvants. Nanocarriers and adjuvants are of particular interest to the fields of vaccines and immunotherapy as they allow for targeted delivery of the antigens and direct the immune response against these antigens in desirable direction (i.e., to either enhance immunogenicity or induce tolerance). Recently, nanoparticles gained particular attention as antigen carriers and adjuvants. This review focuses on a particular subclass of nanoparticles, which are made of nucleic acids, so-called nucleic acid nanoparticles or NANPs. Immunological properties of these novel materials and considerations for their clinical translation are discussed.