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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.)

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.)

The earliest example of in vivo expression of exogenous mRNA is by direct intramuscular injection in mice without the aid of a delivery vehicle. The current state of the art for therapeutic nucleic acid delivery is lipid nanoparticles (LNP), which are composed of cholesterol, a helper lipid, a PEGylated lipid and an ionizable amine-containing lipid. The liver is the primary organ of LNP accumulation following intravenous administration and is also observed to varying degrees following intramuscular and subcutaneous routes. Delivery of nucleic acid to hepatocytes by LNP has therapeutic potential, but there are many disease indications that would benefit from non-hepatic LNP tissue and cell population targeting, such as cancer, and neurological, cardiovascular and infectious diseases. This review will concentrate on the current efforts to develop the next generation of tissue-targeted LNP constructs for therapeutic nucleic acids.

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., (© 2020 Wiley Periodicals LLC.)

The chloroquine family of antimalarials has a long history of use, spanning many decades. Despite this extensive clinical experience, novel applications, including use in autoimmune disorders, infectious disease, and cancer, have only recently been identified. While short term use of chloroquine or hydroxychloroquine is safe at traditional therapeutic doses in patients without predisposing conditions, administration of higher doses and for longer durations are associated with toxicity, including retinotoxicity. Additional liabilities of these medications include pharmacokinetic profiles that require extended dosing to achieve therapeutic tissue concentrations. To improve chloroquine therapy, researchers have turned toward nanomedicine reformulation of chloroquine and hydroxychloroquine to increase exposure of target tissues relative to off-target tissues, thereby improving the therapeutic index. This review highlights these reformulation efforts to date, identifying issues in experimental designs leading to ambiguity regarding the nanoformulation improvements and lack of thorough pharmacokinetics and safety evaluation. Gaps in our current understanding of these formulations, as well as recommendations for future formulation efforts, are presented.

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., Competing Interests: The authors declare no conflicts of interest.

The pharmacokinetics of nanomedicines are complicated by the unique dispositional characteristics of the drug carrier. Most simplistically, the carrier could be a solubilizing platform that allows administration of a hydrophobic drug. Alternatively, the carrier could be stable and release the drug in a controlled manner, allowing for distribution of the carrier to influence distribution of the encapsulated drug. A third potential dispositional mechanism is carriers that are not stably complexed to the drug, but rather bind the drug in a dynamic equilibrium, similar to the binding of unbound drug to protein; since the nanocarrier has distributional and binding characteristics unlike plasma proteins, the equilibrium binding of drug to a nanocarrier can affect pharmacokinetics in unexpected ways, diverging from classical protein binding paradigms. The recently developed stable isotope tracer ultrafiltration assay (SITUA) for nanomedicine fractionation is uniquely suited for distinguishing and comparing these carrier/drug interactions. Here we present the the encapsulated, unencapsulated, and unbound drug fraction pharmacokinetic profiles in rats for marketed nanomedicines, representing examples of controlled release (doxorubicin liposomes, Doxil; and doxorubicin HCl liposome generic), equilibrium binding (paclitaxel cremophor micelle solution, Taxol generic), and solubilizing (paclitaxel albumin nanoparticle, Abraxane; and paclitaxel polylactic acid micelle, Genexol-PM) nanomedicine formulations. The utility of the SITUA method in differentiating these unique pharmacokinetic profiles and its potential for use in establishing generic nanomedicine bioequivalence are discussed., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

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.

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.

To guide developers of innovative and generic drug products that contain nanomaterials, the U.S. Food and Drug Administration issued the draft guidance for industry titled: "Drug Products, Including Biological Products, that Contain Nanomaterials" in December 2017. During the AAPS Guidance Forum on September 11, 2018, participants from industry, academia, and regulatory bodies discussed this draft guidance in an open setting. Two questions raised by the AAPS membership were discussed in more detail: what is the appropriate regulatory pathway for approval of drug products containing nanomaterials, and how to determine critical quality attributes (CQAs) for nanomaterials? During the meeting, clarification was provided on how the new FDA center-led guidance relates to older, specific nanomaterial class, or specific product-related guidances. The lively discussions concluded with some clear observations and recommendations: (I) Important lessons can be learned from how CQAs were determined for, e.g., biologics. (II) Publication of ongoing scientific discussions on strategies and studies determining CQAs of drug products containing nanomaterials will significantly strengthen the science base on this topic. Furthermore, (III) alignment on a global level on how to address new questions regarding nanomedicine development protocols will add to efficient development and approval of these much needed candidate nanomedicines (innovative and generic). Public meetings such as the AAPS Guidance Forum may serve as the place to have these discussions.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Continued advancements in nanotechnology are expanding the boundaries of medical research, most notably as drug delivery agents for treatment against cancer. Drug delivery with nanotechnology can offer greater control over the biodistribution of therapeutic agents to improve the therapeutic index. In the last 20 years, a number of nanomedicines have transitioned into the clinic. As nanomedicines evolve, techniques to properly evaluate their safety and efficacy must also evolve. Characterization methods for nano-based materials must be adapted to the demands of nanomedicine developers and regulators. This second edition book provides updated characterization protocols designed to address the clinical potential of nanomedicines during their preclinical development. In this chapter, the characterization challenges of nanoparticles intended for drug delivery will be discussed, along with examples of advancements and improvements in nanomedicine characterization.

Genomic analyses from patients with cancer have improved the understanding of the genetic elements that drive the disease, provided new targets for treating this relentless disease, and offered criteria for stratifying patient populations that will benefit most from treatments. In the last decade, several new targeted therapies have been approved by the FDA based on these omics findings, leading to significantly improved survival and quality of life for select patient populations. However, many of these precision medicines, e.g., nucleic acid-based therapies and antibodies, suffer from poor plasma stability, suboptimal pharmacokinetic properties, and immunological toxicities that prohibit their clinical translation. Nanotechnology is being explored as a delivery platform that can enable the successful delivery of these precision medicine treatments without these limitations. These precision nanomedicines are able to protect the cargo from degradation or premature/burst release prior to accumulation at the tumor site and improve the selectivity to cancer cells by incorporating ligands that can target receptors overexpressed on the cancer cell surface. Here, we review the development of several precision nanomedicines based on genomic analysis of clinical samples, actively targeted nanoparticle delivery systems in the clinic, and the pathophysiological barriers of the tumor microenvironment. Successful translation of these precision nanomedicine initiatives will require an effective collaboration between basic and clinical investigators to match the right patient with the right therapies and to deliver them at therapeutic concentrations which will improve overall treatment responses.

Zeta potential is often used to approximate a nanoparticle's surface charge, i.e., cationic, anionic, or neutral character, and has become a standard characterization technique to evaluate nanoparticle surfaces. While useful, zeta potential values provide only very general conclusions about surface charge character. Without a thorough understanding of the measurement parameters and limitations of the technique, these values can become meaningless. This case study attempts to explore the sensitivity of zeta potential measurement using specifically formulated cationic, anionic, and neutral liposomes. This study examines zeta potential dependence on pH and ionic strength, resolving power, and highlights the sensitivity of zeta potential to charged liposomes. Liposomes were prepared with cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and varying amounts of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS). A strong linear relationship was noted between zeta potential values and the mole percentage of charged lipids within a liposome (e.g., cationic DOTAP or anionic DOPS). This finding could be used to formulate similar liposomes to a specific zeta potential, potentially of importance for systems sensitive to highly charged species. In addition, cationic and anionic liposomes were titrated with up to two mole percent of the neutral lipid 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (lipid-PEG; LP). Very small amounts of the lipid-PEG (<0.2 mol%) were found to impart stability to the DOTAP- and DOPS-containing liposomes without significantly affecting other physicochemical properties of the formulation, providing a simple approach to making stable liposomes with cationic and anionic surface charge.

The vast majority of drug product candidates in early development fail to progress to clinics. This is true for products containing nanomaterials just as for other types of pharmaceuticals. Early development pathways should therefore place high priority on experiments that help candidates fail faster and less expensively. Nanomedicines fail for many reasons, but some are more avoidable than others. Some of the points of failure are not considerations in the development of small molecules or biopharmaceuticals, and so may be unexpected, even to those with previous experience bringing drug products to the clinic. This article reviews experiments that have proven useful in providing "go/no-go" decision-making data for nanomedicines in early preclinical development. Of course, the specifics depend on the particulars of the drug product and the nanomaterial type, and not every product shares the same development pathway or the same potential points of failure. Here, we focus on challenges that differ from those in the development of traditional small molecule therapeutics, and on experiments that reveal deficiencies that can only be corrected by essentially starting over-altering the nanomedicine to an extent that all previous characterization and proof-of-concept testing must be repeated. Conducting these experiments early in the development process can save significant resources and time and allow developers to focus on derisked candidates with a greater likelihood of ultimate success.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer- related deaths. PDAC remains one of the most difficult-to-treat cancers, owing to its unique pathobiological features: a nearly impenetrable desmoplastic stroma, and hypovascular and hypoperfused tumour vessels render most treatment options largely ineffective. Progress in understanding the pathobiology and signalling pathways involved in disease progression is helping researchers to develop novel ways to fight PDAC, including improved nanotechnology-based drug-delivery platforms that have the potential to overcome the biological barriers of the disease that underlie persistent drug resistance. So-called 'nanomedicine' strategies have the potential to enable targeting of the Hedgehog-signalling pathway, the autophagy pathway, and specific RAS-mutant phenotypes, among other pathological processes of the disease. These novel therapies, alone or in combination with agents designed to disrupt the pathobiological barriers of the disease, could result in superior treatments, with increased efficacy and reduced off-target toxicities compared with the current standard-of-care regimens. By overcoming drug-delivery challenges, advances can be made in the treatment of PDAC, a disease for which limited improvement in overall survival has been achieved over the past several decades. We discuss the approaches to nanomedicine that have been pursued to date and those that are the focus of ongoing research, and outline their potential, as well as the key challenges that must be overcome.

Polyethylene glycol (PEG) is an important tool for increasing the biocompatibility of nanoparticle therapeutics. Understanding how these potential nanomedicines will react after they have been introduced into the bloodstream is a critical component of the preclinical evaluation process. Hence, it is paramount that better methods for separating, characterizing, and analyzing these complex and polydisperse formulations are developed. We present a method for separating nominal 30-nm gold nanoparticles coated with various molecular weight PEG moieties that uses only phosphate-buffered saline as the mobile phase, without the need for stabilizing surfactants. The optimized asymmetric-flow field-flow fractionation technique using in-line multiangle light scattering, dynamic light scattering, refractive index, and UV-vis detectors allowed successful separation and detection of a mixture of nanoparticles coated with 2-, 5-, 10-, and 20-kDa PEG. The particles coated with the larger PEG species (10 and 20 kDa) were eluted at times significantly earlier than predicted by field-flow fractionation theory. This was attributed to a lower-density PEG shell for the higher molecular weight PEGylated nanoparticles, which allows a more fluid PEG surface that can be greater influenced by external forces. Hence, the apparent particle hydrodynamic size may fluctuate significantly depending on the overall density of the stabilizing surface coating when an external force is applied. This has considerable implications for PEGylated nanoparticles intended for in vivo application, as nanoparticle size is important for determining circulation times, accumulation sites, and routes of excretion, and highlights the importance and value of the use of secondary size detectors when one is working with complex samples in asymmetric-flow field-flow fractionation.

Surface characteristics of a nanoparticle, such as functionalization with polyethylene glycol (PEG), are critical to understand and achieve optimal biocompatibility. Routine physicochemical characterization such as UV-vis spectroscopy (for gold nanoparticles), dynamic light scattering, and zeta potential are commonly used to assess the presence of PEG. However, these techniques are merely qualitative and are not sensitive enough to distinguish differences in PEG quantity, density, or presentation. As an alternative, two methods are described here which allow for quantitative measurement of PEG on PEGylated gold nanoparticles. The first, a displacement method, utilizes dithiothreitol to displace PEG from the gold surface. The dithiothreitol-coated gold nanoparticles are separated from the mixture via centrifugation, and the excess dithiothreitol and dissociated PEG are separated through reversed-phase high-performance liquid chromatography (RP-HPLC). The second, a dissolution method, utilizes potassium cyanide to dissolve the gold nanoparticles and liberate PEG. Excess CN(-), Au(CN)2 (-), and free PEG are separated using RP-HPLC. In both techniques, the free PEG can be quantified against a standard curve using charged aerosol detection. The displacement and dissolution methods are validated here using 2-, 5-, 10-, and 20-kDa PEGylated 30-nm colloidal gold nanoparticles. Further value in these techniques is demonstrated not only by quantitating the total PEG fraction but also by being able to be adapted to quantitate the free unbound PEG and the bound PEG fractions. This is an important distinction, as differences in the bound and unbound PEG fractions can affect biocompatibility, which would not be detected in techniques that only quantitate the total PEG fraction.

Proteins bound to nanoparticle surfaces are known to affect particle clearance by influencing immune cell uptake and distribution to the organs of the mononuclear phagocytic system. The composition of the protein corona has been described for several types of nanomaterials, but the role of the corona in nanoparticle biocompatibility is not well established. In this study we investigate the role of nanoparticle surface properties (PEGylation) and incubation times on the protein coronas of colloidal gold nanoparticles. While neither incubation time nor PEG molecular weight affected the specific proteins in the protein corona, the total amount of protein binding was governed by the molecular weight of PEG coating. Furthermore, the composition of the protein corona did not correlate with nanoparticle hematocompatibility. Specialized hematological tests should be used to deduce nanoparticle hematotoxicity. From the clinical editor: It is overall unclear how the protein corona associated with colloidal gold nanoparticles may influence hematotoxicity. This study warns that PEGylation itself may be insufficient, because composition of the protein corona does not directly correlate with nanoparticle hematocompatibility. The authors suggest that specialized hematological tests must be used to deduce nanoparticle hematotoxicity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Aim: Disseminated intravascular coagulation is an increasing concern for certain types of engineered nanomaterials. Recent studies have shed some light on the nanoparticle physicochemical properties contributing to this toxicity; however, the mechanisms are poorly understood. Leukocyte procoagulant activity (PCA) is a key factor contributing to the initiation of this toxicity. We have previously reported on the exaggeration of endotoxin-induced PCA by cationic dendrimers. Herein, we report an effort to discern the mechanism., Materials & Methods: Poly(amidoamine) dendrimers with various sizes and surface functionalities were studied in vitro by the recalcification test, flow cytometry and other relevant assays., Results & Conclusion: Cationic dendrimers exaggerated endotoxin-induced PCA, but their anionic or neutral counterparts did not; the cationic charge prompts this phenomenon, but different cationic surface chemistries do not influence it. Cationic dendrimers and endotoxin differentially affect the PCA complex. The inhibition of phosphoinositol 3 kinase by dendrimers contributes to the exaggeration of the endotoxin-induced PCA.

The Nanotechnology Characterization Laboratory's (NCL) unique set-up has allowed our lab to handle and test a variety of nanoparticle platforms intended for the delivery of cancer therapeutics and/or imaging contrast agents. Over the last six years, the NCL has characterized more than 250 different nanomaterials from more than 75 different investigators. These submitted nanomaterials stem from a range of backgrounds and experiences, including government, academia and industry. This has given the NCL a unique and valuable opportunity to observe trends in nanoparticle safety and biocompatibility, as well as note some of the common mistakes and oversights of nanoformulation. While not exhaustive, this article aims to share some of the most common pitfalls observed by the NCL as they relate to nanoparticle synthesis, purification, characterization and analysis.

Expression of a retroviral Gag protein in mammalian cells leads to the assembly of virus particles. In vitro, recombinant Gag proteins are soluble but assemble into virus-like particles (VLPs) upon addition of nucleic acid. We have proposed that Gag undergoes a conformational change when it is at a high local concentration and that this change is an essential prerequisite for particle assembly; perhaps one way that this condition can be fulfilled is by the cooperative binding of Gag molecules to nucleic acid. We have now characterized the assembly in human cells of HIV-1 Gag molecules with a variety of defects, including (i) inability to bind to the plasma membrane, (ii) near-total inability of their capsid domains to engage in dimeric interaction, and (iii) drastically compromised ability to bind RNA. We find that Gag molecules with any one of these defects still retain some ability to assemble into roughly spherical objects with roughly correct radius of curvature. However, combination of any two of the defects completely destroys this capability. The results suggest that these three functions are somewhat redundant with respect to their contribution to particle assembly. We suggest that they are alternative mechanisms for the initial concentration of Gag molecules; under our experimental conditions, any two of the three is sufficient to lead to some semblance of correct assembly.

The study of the potential risks associated with the manufacture, use, and disposal of nanoscale materials, and their mechanisms of toxicity, is important for the continued advancement of nanotechnology. Currently, the most widely accepted paradigms of nanomaterial toxicity are oxidative stress and inflammation, but the underlying mechanisms are poorly defined. This review will highlight the significance of autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Most endocytic routes of nanomaterial cell uptake converge upon the lysosome, making the lysosomal compartment the most common intracellular site of nanoparticle sequestration and degradation. In addition to the endo-lysosomal pathway, recent evidence suggests that some nanomaterials can also induce autophagy. Among the many physiological functions, the lysosome, by way of the autophagy (macroautophagy) pathway, degrades intracellular pathogens, and damaged organelles and proteins. Thus, autophagy induction by nanoparticles may be an attempt to degrade what is perceived by the cell as foreign or aberrant. While the autophagy and endo-lysosomal pathways have the potential to influence the disposition of nanomaterials, there is also a growing body of literature suggesting that biopersistent nanomaterials can, in turn, negatively impact these pathways. Indeed, there is ample evidence that biopersistent nanomaterials can cause autophagy and lysosomal dysfunctions resulting in toxicological consequences.

Expression of a retroviral protein, Gag, in mammalian cells is sufficient for assembly of immature virus-like particles (VLPs). VLP assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We have investigated the role of SP1, a spacer between CA and NC in HIV-1 Gag, in VLP assembly. Mutational analysis showed that even subtle changes in the first 4 residues of SP1 destroy the ability of Gag to assemble correctly, frequently leading to formation of tubes or other misassembled structures rather than proper VLPs. We also studied the conformation of the CA-SP1 junction region in solution, using both molecular dynamics simulations and circular dichroism. Consonant with nuclear magnetic resonance (NMR) studies from other laboratories, we found that SP1 is nearly unstructured in aqueous solution but undergoes a concerted change to an α-helical conformation when the polarity of the environment is reduced by addition of dimethyl sulfoxide (DMSO), trifluoroethanol, or ethanol. Remarkably, such a coil-to-helix transition is also recapitulated in an aqueous medium at high peptide concentrations. The exquisite sensitivity of SP1 to mutational changes and its ability to undergo a concentration-dependent structural transition raise the possibility that SP1 could act as a molecular switch to prime HIV-1 Gag for VLP assembly. We suggest that changes in the local environment of SP1 when Gag oligomerizes on nucleic acid might trigger this switch.

The binding of retinoic acid to mutants of Cellular Retinoic Acid Binding Protein II (CRABPII) was evaluated to better understand the importance of the direct protein/ligand interactions. The important role of Arg111 for the correct structure and function of the protein was verified and other residues that directly affect retinoic acid binding have been identified. Furthermore, retinoic acid binding to CRABPII mutants that lack all previously identified interacting amino acids was rescued by providing a carboxylic acid dimer partner in the form of a Glu residue., (2008 Wiley-Liss, Inc.)

Expression of the retroviral Gag protein leads to formation of virus-like particles in mammalian cells. In vitro and in vivo experiments show that nucleic acid is also required for particle assembly. However, several studies have demonstrated that chimeric proteins in which the nucleocapsid domain of Gag is replaced by a leucine zipper motif can also assemble efficiently in mammalian cells. We have now analyzed assembly by chimeric proteins in which nucleocapsid of human immunodeficiency virus type 1 (HIV-1) Gag is replaced by either a dimerizing or a trimerizing zipper. Both proteins assemble well in human 293T cells; the released particles lack detectable RNA. The proteins can coassemble into particles together with full-length, wild-type Gag. We purified these proteins from bacterial lysates. These recombinant "Gag-Zipper" proteins are oligomeric in solution and do not assemble unless cofactors are added; either nucleic acid or inositol phosphates (IPs) can promote particle assembly. When mixed with one equivalent of IPs (which do not support assembly of wild-type Gag), the "dimerizing" Gag-Zipper protein misassembles into very small particles, while the "trimerizing" protein assembles correctly. However, addition of both IPs and nucleic acid leads to correct assembly of all three proteins; the "dimerizing" Gag-Zipper protein also assembles correctly if inositol hexakisphosphate is supplemented with other polyanions. We suggest that correct assembly requires both oligomeric association at the C terminus of Gag and neutralization of positive charges near its N terminus.

Rational redesign of the binding pocket of Cellular Retinoic Acid Binding Protein II (CRABPII) has provided a mutant that can bind retinal as a protonated Schiff base, mimicking the binding observed in rhodopsin. The reengineering was accomplished through a series of choreographed manipulations to ultimately orient the reactive species (the epsilon-amino group of Lys132 and the carbonyl of retinal) in the proper geometry for imine formation. The guiding principle was to achieve the appropriate Bürgi-Dunitz trajectory for the reaction to ensue. Through crystallographic analysis of protein mutants incapable of forming the requisite Schiff base, a highly ordered water molecule was identified as a key culprit in orienting retinal in a nonconstructive manner. Removal of the ordered water, along with placing reinforcing mutations to favor the desired orientation of retinal, led to a triple mutant CRABPII protein capable of nanomolar binding of retinal as a protonated Schiff base. The high-resolution crystal structure of all-trans-retinal bound to the CRABPII triple mutant (1.2 A resolution) unequivocally illustrates the imine formed between retinal and the protein.

A single multi-domain viral protein, termed Gag, is sufficient for assembly of retrovirus-like particles in mammalian cells. We have purified the human immunodeficiency virus type 1 (HIV-1) Gag protein (lacking myristate at its N terminus and the p6 domain at its C terminus) from bacteria. This protein is capable of assembly into virus-like particles in a defined in vitro system. We have reported that it is in monomer-dimer equilibrium in solution, and have described a mutant Gag protein that remains monomeric at high concentrations in solution. We report that the mutant protein retains several properties of wild-type Gag. This mutant enabled us to analyze solutions of monomeric protein. Hydrodynamic studies on the mutant protein showed that it is highly asymmetric, with a frictional ratio of 1.66. Small-angle neutron scattering (SANS) experiments confirmed its asymmetry and yielded an R(g) value of 34 A. Atomic-level structures of individual domains within Gag have previously been determined, but these domains are connected in Gag by flexible linkers. We constructed a series of models of the mutant Gag protein based on these domain structures, and tested each model computationally for its agreement with the experimental hydrodynamic and SANS data. The only models consistent with the data were those in which Gag was folded over, with its N-terminal matrix domain near its C-terminal nucleocapsid domain in three-dimensional space. Since Gag is a rod-shaped molecule in the assembled immature virion, these findings imply that Gag undergoes a major conformational change upon virus assembly.

Due to the difficulties in handling and manipulating membrane-bound proteins, such as rhodopsin, and the lack of crystallographic information on the cone opsins, we have opted to engineer a protein mimic of the transmembrane G-protein coupled receptor. Human cellular retinoic acid binding protein (CRABPII), a well studied and characterized protein, has been reengineered into a protein that now will bind retinal as a protonated Schiff base with high binding affinity (Kd = 2 nM) mimicking that of rhodopsin.

A study of intramolecular hydrogen bonding in chloroform for a small combinatorial library of nine triamides with varying connecting chain length has been completed. The starting materials for the triamides are three diacids (succinic, glutaric, and adipic acid) and three amino acids (glycine, beta-alanine, and gamma-aminobutyric acid). The preferences for the head-to-tail type of folding pattern are identified for the smaller triamides (1 and 4). The preference for the head-to-tail folding pattern can be explained by the energetic superiority of an optimal hydrogen bond geometry in which the NH---O bond angle is near linearity. The beta-alanine containing triamides 2, 5, and 8 are resistant to intramolecular hydrogen bonding, especially to nearest neighbor hydrogen bonding. At lower temperatures, triamides 2 and 5 exhibit a small population of head-to-tail type of folding, while triamide 8 shows a significant population of bifurcated conformation. Triamide 6, 7, and 9 prefer bicyclic structures involving nearest neighbor hydrogen bonding. A nine-membered ring is large enough to accommodate a near linear N-H--O bond angle. Entropic effects are probably responsible for the preference of the nine-membered ring over a 12- or a 14-membered ring. The enhancement of hydrogen bonding in triamide 9 is enormous, and both NHs have a very large temperature dependence of chemical shifts (-15 ppb/K and -13.3 ppb/K for the terminal and the internal NH protons, respectively). Using appropriate temperature-dependent lower and upper limits of chemical shifts, a van't Hoff analysis gives the hydrogen bond strength for the terminal NH (DeltaH = -3.1 +/- 0.5 kcal/mol) and for the internal NH (DeltaH = -2.8 +/- 0.5 kcal/mol). The increased hydrogen bond strength is taken as evidence for hydrogen-bonding cooperativity from the two mutually enhanced individual hydrogen bonds. A near linear NH--O bond angle is required for this effect.