Advancements in Nanoparticle Characterization.

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.

Assessment of Protein Binding Using Asymmetric-Flow Field-Flow Fractionation Combined with Multi-angle Light Scattering and Dynamic Light Scattering.

Asymmetric-flow field-flow fractionation (AF4) is a valuable tool to separate and assess different size populations in nanotherapeutics. When coupled with both static light scattering and dynamic light scattering, it can be used to qualitatively assess protein binding to nanoparticles by comparing the shape factors for both non-plasma-incubated samples and plasma-incubated samples. The shape factor is defined as the ratio of the derived root mean square radius (by static light scattering) to the measured hydrodynamic radius (by dynamic light scattering). The shape factor gives an idea of where the center of mass lies in a nanoparticle, and any shift in the shape factor to larger values is indicative of a mass addition to the periphery of the nanoparticle and suggests the presence of protein binding. This protocol will discuss how to set up an experiment to assess protein binding in nanoparticles using AF4, multi-angle light scattering (MALS), and dynamic light scattering (DLS).

Quantitation of Active Pharmaceutical Ingredients in Polymeric Drug Products Using Elemental Analysis.

Polymeric prodrugs have gained significant popularity as a strategy to enhance the bioavailability and improve the pharmacokinetic properties of active pharmaceutical ingredients (API). Since the amount of the API in a polymeric prodrug product directly impacts both safety and efficacy, there is a pressing need for robust and accurate analytical methods to quantify the API in these formulations. Presently, drug quantification methods include reversed-phase high-performance liquid chromatography (RP-HPLC) and size exclusion chromatography (SEC)-based molecular weight determination. Even though these methods are highly precise and reproducible, a deep understanding of chromatography is required for complex method development, including optimization of the elution profile and selecting the appropriate column and mobile phase. In this chapter, we introduce the automated elemental analyzer for drug quantification, which is simple to use and does not require special method development.

Measuring Size and Number Concentration of Metallic Nanoparticles Using spICP-MS.

This protocol describes how to measure the size and concentration of individual metallic nanoparticles using inductively coupled plasma-mass spectrometry (ICP-MS) in single-particle (sp) mode. Accurately determining the size of individual nanoparticles on a per-particle basis, both quickly and accurately, is an ever-increasing need within nanoparticle characterization. ICP-MS is capable of measuring a broad range of metallic nanoparticle sizes with high resolution, thus allowing the measurement of multiple particle populations for the quality assessment of nanoformulations. Additionally, spICP-MS can accurately determine particle concentrations without the need for concentration standards.

Nanoparticle Size Distribution and Stability Assessment Using Asymmetric-Flow Field-Flow Fractionation.

Nanomaterials are inherently polydisperse. Traditional techniques, such as the widely used batch-mode dynamic light-scattering (DLS) analysis, are not ideal nor thoroughly descriptive enough to define the full complexity of these materials. Asymmetric-flow field-flow fractionation (AF4) with various in-line detectors, such as ultraviolet-visible (UV-vis), multi-angle light scattering (MALS), refractive index (RI), and DLS, is an alternative technique that can provide flow-mode analysis of not only size distribution but also shape, drug release/stability, and protein binding.

A Static Headspace Gas Chromatography Method for Quantitation of Residual Solvents in Nanoformulations.

Various organic solvents are widely used in the manufacturing, processing, and purification of drug substances, drug products, formulations, excipients, etc. These solvents must be removed to the lowest amount permitted, as they do not possess any therapeutic advantages and may cause undesirable toxicities. Therefore, a rapid and sensitive analytical method for the quantitation of residual solvents is needed. The following chapter presents a static headspace gas chromatographic (HSGC) method for determining the concentration of common residual solvents in various nanoformulations. An efficient and sensitive HSGC method has been developed using PerkinElmer's headspace autosampler/gas chromatographic system with a flame ionization detector (FID) and validated according to the International Conference for Harmonization (ICH) guideline Q3C. The method validation indicates that the method is specific, linear, accurate, precise, and sensitive for the analyzed solvents. The method is suitable for the analysis of 13 residual solvents (methanol, ethanol, acetone, diethyl ether, 2-propanol, acetonitrile, 1-propanol, ethyl acetate, tetrahydrofuran, dichloromethane, chloroform, 1-butanol, and pyridine) and utilizes an Elite 624 Crossbond 6% cyanopropylphenyl, 94% dimethylpolysiloxanes column with helium as a carrier gas.

Particle Size and Concentration Measurement Using the Spectradyne nCS1 Instrument.

This protocol describes the use of the Spectradyne nCS1 instrument to measure the particles per mL concentration and size of nanoparticles. The Spectradyne nCS1 is a particle-analyzing instrument that uses microfluidic resistive pulse sensing, rather than optical measurements, to determine the size and concentration of samples. The size and concentration of a sample are determined by measuring the changes in voltage as particles travel through a nano-constriction in the microfluidic cartridge. This method also has the advantage over optical techniques in that measurements are not dependent on the type of material being measured (e.g., refractive index of the sample itself is not needed for accurate analysis), and only microliters (typically 5 µL) of a sample are needed for analysis.

Ion Quantitation in Liposomal Products Using RP-HPLC with Charged Aerosol Detection.

Ion concentration in liposomal drugs is important for drug stability and drug release profile. However, quantifying ion concentration in liposomal drugs is challenging due to the absence of chromophores or fluorophores of ions and the efficiency of their release from the liposome structure. To address these issues, a method based on reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with a charged aerosol detector (CAD) has been developed to determine total, internal, and external ions in drug-loaded liposomal products. In this protocol, we focused on the quantitation of ammonium and sulfate ions in liposomal products, using generic PEGylated liposomal doxorubicin as an example. This method can be extended to calcium, acetate, and other ions in different liposomal formulations with slight modifications.

Multicolor flow cytometry-based immunophenotyping for preclinical characterization of nanotechnology-based formulations: an insight into structure activity relationship and nanoparticle biocompatibility profiles.

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.

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.

Trends and patterns in cancer nanotechnology research: A survey of NCI's caNanoLab and nanotechnology characterization laboratory.

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.

The importance of nanoparticle physicochemical characterization for immunology research: What we learned and what we still need to understand.

Physicochemical characterization of nanoparticles intended for immunology research is important as it helps explain the observed immunological effects. More importantly, it relates the physicochemical properties with the immunological properties to draw meaningful conclusions. There are many physicochemical parameters, with each having numerous analytical techniques and instrumentation to measure them. Thus, where to begin can be challenging even for the experienced scientist. This paper aims to provide guidance to the immunology scientist on how best to characterize their nanoparticles. A step-by-step guide for the physicochemical characterization of liposomal formulations, based on the FDA's guidance for industry for Liposome Drug Products, is provided. Eight critical quality attributes have been identified and for each, the methodology and the physicochemical questions one should consider are discussed. This chapter also addresses common physicochemical characterization mistakes and concludes with a perspective on the type of measurements needed to address current physicochemical characterization gaps and challenges.

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.

Challenges in the development of nanoparticle-based imaging agents: Characterization and biology.

Despite imaging agents being some of the earliest nanomedicines in clinical use, the vast majority of current research and translational activities in the nanomedicine field involves therapeutics, while imaging agents are severely underrepresented. The reasons for this lack of representation are several fold, including difficulties in synthesis and scale-up, biocompatibility issues, lack of suitable tissue/disease selective targeting ligands and receptors, and a high bar for regulatory approval. The recent focus on immunotherapies and personalized medicine, and development of nanoparticle constructs with better tissue distribution and selectivity, provide new opportunities for nanomedicine imaging agent development. This manuscript will provide an overview of trends in imaging nanomedicine characterization and biocompatibility, and new horizons for future development. This article is categorized under: Diagnostic Tools > in vivo Nanodiagnostics and Imaging Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

The utility of asymmetric flow field-flow fractionation for preclinical characterization of nanomedicines.

Dynamic light scattering (DLS), transmission electron microscopy (TEM), and reversed phase-high performance liquid chromatography (RP-HPLC) are staples of nanoparticle characterization for size distribution, shape/morphology, and composition, respectively. These techniques are simple and provide important details on sample characteristics. However, DLS and TEM are routinely done in batch-mode, while RP-HPLC affords separation of components within the entire sample population, regardless of sample polydispersity. While batch-mode analysis is informative and should be a first-step analysis for any material, it may not be ideal for polydisperse formulations, such as many nanomedicines. Herein, we describe the utility of asymmetric flow field-flow fractionation (AF4) as a useful tool for a more thorough understanding of these inherently polydisperse materials. AF4 was coupled with in-line DLS for an enhanced separation and resolution of various size populations in a nanomaterial. Additionally, the various size populations were collected for offline analysis by TEM for an assessment of different shape populations, or RP-HPLC to provide a compositional analysis of each individual size population. This technique was also extended to assess nanoparticle stability, i.e., drug release, both in buffer and physiologically relevant matrix, as well as qualitatively evaluate the protein binding capacity of nanomedicines. Overall, AF4 is proven to be a very versatile technique and can provide a wealth of information on a material's polydispersity and stability. Moreover, the ability to conduct analysis in physiological matrices provides an advantage that many other routine analytical techniques do not. Graphical Abstract.

Sizing up the Next Generation of Nanomedicines.

During the past two decades the nanomedicine field has experienced significant progress. To date, over sixty nanoparticle (NP) formulations have been approved in the US and EU while many others are in clinical or preclinical development, indicating a concerted effort to translate promising bench research to commercially viable pharmaceutical products. The use of NPs as novel drug delivery systems, for example, can improve drug safety and efficacy profiles and enable access to intracellular domains of diseased cells, thus paving the way to previously intractable biological targets. However, the measurement of their physicochemical properties presents substantial challenges relative to conventional injectable formulations. In this perspective, we focus exclusively on particle size, a core property and critical quality attribute of nanomedicines. We present an overview of relevant state-of-the-art technologies for particle sizing, highlighting the main parameters that can influence the selection of techniques suitable for a specific size range or material. We consider the increasing need, and associated challenge, to measure size in physiologically relevant media. We detail the importance of standards, key to validate any measurement, and the need for suitable reference materials for processes used to characterize novel and complex NPs. This perspective highlights issues critical to achieve compliance with regulatory guidelines and to support research and manufacturing quality control.

Total drug quantification in prodrugs using an automated elemental analyzer.

Polymeric prodrugs have become an increasingly popular strategy for improving the pharmacokinetic properties of active pharmaceutical ingredients (API). Therefore, identifying a robust method for quantification of the API in these prodrug products is a key part of the drug development process. Current drug quantification methods include hydrolysis followed by reversed phase high-performance liquid chromatography (RP-HPLC), size exclusion chromatography (SEC)-based molecular weight determination, and mass spectrometry. These methods tend to be time-consuming and often require challenging method development. Here, we present a comparative study highlighting the automated elemental analyzer as a facile approach to drug quantification in this up-and-coming class of therapeutics. A polymeric prodrug using poly(L-lysine succinylated) (PLS) and the drug lamivudine (LAM) was prepared and analyzed using the elemental analyzer in comparison to the traditional approaches of hydrolysis followed by RP-HPLC and SEC using multi-angle light scattering (MALS) detection. The elemental analysis approach showed excellent agreement with the conventional methods but proved much less laborious, highlighting this as a rapid and sensitive analytical method for the quantitative determination of drug loading in polymeric prodrug products.

Quantitative biophysical analysis defines key components modulating recruitment of the GTPase KRAS to the plasma membrane.

The gene encoding the GTPase KRAS is frequently mutated in pancreatic, lung, and colorectal cancers. The KRAS fraction in the plasma membrane (PM) correlates with activation of the mitogen-activated protein kinase (MAPK) pathway and subsequent cellular proliferation. Understanding KRAS's interaction with the PM is challenging given the complexity of the cellular environment. To gain insight into key components necessary for KRAS signal transduction at the PM, we used synthetic membranes such as liposomes and giant unilamellar vesicles. Using surface plasmon resonance (SPR) spectroscopy, we demonstrated that KRAS and Raf-1 proto-oncogene Ser/Thr kinase (RAF1) domains interact with these membranes primarily through electrostatic interactions with negatively charged lipids reinforced by additional interactions involving phosphatidyl ethanolamine and cholesterol. We found that the RAF1 region spanning RBD through CRD (RBDCRD) interacts with the membrane significantly more strongly than the isolated RBD or CRD domains and synergizes KRAS partitioning to the membrane. We also found that calmodulin and phosphodiesterase 6 delta (PDE6δ), but not galectin3 previously proposed to directly interact with KRAS, passively sequester KRAS and prevent it from partitioning into the PM. RAF1 RBDCRD interacted with membranes preferentially at nonraft lipid domains. Moreover, a C-terminal O-methylation was crucial for KRAS membrane localization. These results contribute to a better understanding of how the KRAS-membrane interaction is tuned by multiple factors whose identification could inform drug discovery efforts to disrupt this critical interaction in diseases such as cancer.

Ion quantification in liposomal drug products using high performance liquid chromatography.

A simple, straightforward analytical method based on liquid chromatography has been optimized to quantify total, internal, and external ions in drug-loaded liposomal products. The quantification of ammonium and sulfate ions in Doxil is detailed; although, the methodology has been extrapolated to quantitate a variety of ions, including calcium, acetate, and others in several different liposomal formulations. Total ion concentrations were measured after disruption of the liposome via lyophilization, to liberate all components. External ion concentrations were made following membrane centrifugation, without disruption of the liposome structure, where the permeate fraction was analyzed for external ion quantities. The internal ion fraction was derived from mass balance of the total and external ion measurements. High performance liquid chromatography (HPLC), equipped with different separation columns, and coupled to a charged aerosol detector, was employed for all ion quantifications. The analytical measurements were confirmed using simple stoichiometry based on the drug crystallization of doxorubicin within the liposome interior. The method presented herein is quick, highly accurate, and has significantly improved lower limits of detection and quantification over other traditional methods. As more follow-on versions of Doxil are being developed, this facile approach to ion quantitation can be used to help establish compositional similarity to the reference listed drug.

Feraheme® suppresses immune function of human T lymphocytes through mitochondrial damage and mitoROS production.

Despite attractive properties for both therapeutic and diagnostic applications, the clinical use of iron oxide nanoparticles (IONPs) is limited to iron replacement in severely anemic patient populations. While several studies have reported about the immunotoxicity of IONPs, the mechanisms of this toxicity are mostly unknown. We conducted a mechanistic investigation using an injectable form of IONP, Feraheme®. In the cultures of primary human T cells, Feraheme induced miotochondrial oxidative stress and resulted in changes in mitochondrial dynamics, architecture, and membrane potential. These molecular events were responsible for the decrease in cytokine production and proliferation of mitogen-activated T cells. The induction of mitoROS by T cells in response to Feraheme was insufficient to induce total redox imbalance at the cellular level. Consequently, we resolved this toxicity by the addition of the mitochondria-specific antioxidant MitoTEMPO. We further used these findings to develop an experimental framework consisting of critical assays that can be used to estimate IONP immunotoxicity. We explored this framework using several immortalized T-cell lines and found that none of them recapitulate the toxicity observed in the primary cells. Next, we compared the immunotoxicity of Feraheme to that of other FDA-approved iron-containing complex drug formulations and found that the mitochondrial damage and the resulting suppression of T-cell function are specific to Feraheme. The framework, therefore, can be used for comparing the immunotoxicity of Feraheme with that of its generic versions, while other iron-based complex drugs require case-specific mechanistic investigation.