Regulatory Science Perspective on the Analysis of Microplastics and Nanoplastics in Human Food.

Microplastics are increasingly reported, not only in the environment but also in a wide range of food commodities. While studies on microplastics in food abound, the current state of science is limited in its application to regulatory risk assessment by a continued lack of standardized definitions, reference materials, sample collection and preparation procedures, fit-for purpose analytical methods for real-world and environmentally relevant plastic mixtures, and appropriate quality controls. This is particularly the case for nanoplastics. These methodological challenges hinder robust, quantitative exposure assessments of microplastic and nanoplastic mixtures from food consumption. Furthermore, limited toxicological studies on whether microplastics and nanoplastics adversely impact human health are also impeded by methodology challenges. Food safety regulatory agencies must consider both the exposure and the risk of contaminants of emerging concern to ascertain potential harm. Foundational to this effort is access to and application of analytical methods with the capability to quantify and characterize micro- and nanoscale sized polymers in complex food matrices. However, the early stages of method development and application of early stage methods to study the distribution and potential health effects of microplastics and nanoplastics in food have largely been done without consideration of the stringent requirements of methods to inform regulatory activities. We provide regulatory science perspectives on the state of knowledge regarding the occurrence of microplastics and nanoplastics in food and present our general approach for developing, validating, and implementing analytical methods for regulatory purposes.

Optimization of Detection of Gadodiamide Brain Retention in Rats Using Quantitative T2 Mapping and Intraperitoneal Administration.

BACKGROUND: Currently, the gadolinium retention in the brain after the use of contrast agents is studied by T1 -weighted magnetic resonance imaging (MRI) (T1 w) and T1 mapping. The former does not provide easily quantifiable data and the latter requires prolonged scanning and is sensitive to motion. T2 mapping may provide an alternative approach. Animal studies of gadolinium retention are complicated by repeated intravenous (IV) dosing, whereas intraperitoneal (IP) injections might be sufficient. HYPOTHESIS: T2 mapping will detect the changes in the rat brain due to gadolinium retention, and IP administration is equivalent to IV for long-term studies. STUDY TYPE: Prospective longitudinal. ANIMAL MODEL: A total of 31 Sprague-Dawley rats administered gadodiamide IV (N = 8) or IP (N = 8), or saline IV (N = 6) or IP (N = 9) 4 days per week for 5 weeks. FIELD STRENGTH/SEQUENCES: A 7 T, T1 w, and T2 mapping. ASSESSMENT: T2 relaxation and image intensities in the deep cerebellar nuclei were measured pre-treatment and weekly for 5 weeks. Then brains were assessed for neuropathology (N = 4) or gadolinium content using inductively coupled plasma mass spectrometry (ICP-MS, N = 12). STATISTICAL TESTS: Repeated measures analysis of variance with post hoc Student-Newman-Keuls tests and Hedges' effect size. RESULTS: Gadolinium was detected by both approaches; however, T2 mapping was more sensitive (effect size 2.32 for T2 vs. 0.95 for T1 w), and earlier detection (week 3 for T2 vs. week 4 for T1 w). ICP-MS confirmed the presence of gadolinium (3.076 ± 0.909 nmol/g in the IV group and 3.948 ± 0.806 nmol/g in the IP group). There was no significant difference between IP and IV groups (ICP-MS, P = 0.109; MRI, P = 0.696). No histopathological abnormalities were detected in any studied animal. CONCLUSION: T2 relaxometry detects gadolinium retention in the rat brain after multiple doses of gadodiamide irrespective of the route of administration. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 1.

Sierpinski Pyramids by Molecular Entanglement.

Planar, terpyridine-based metal complexes with the Sierpinski triangular motif and alkylated corners undergo a second self-assembly event to give megastructural Sierpinski pyramids; assembly is driven by the facile lipophilic-lipophilic association of the alkyl moieties and complementary perfect fit of the triangular building blocks. Confirmation of the 3D, pyramidal structures was verified and supported by a combination of TEM, AFM, and multiscale simulation techniques.

Protein corona composition does not accurately predict hematocompatibility of colloidal gold nanoparticles.

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.

Comprehensive analysis of common polymers using hyphenated TGA-FTIR-GC/MS and Raman spectroscopy towards a database for micro- and nanoplastics identification, characterization, and quantitation.

Environmental contamination by micro- and nanoplastics (MNPs) is well documented with potential for their increased accumulation globally. Growing public concern over environmental, ecological, and human exposure to MNPs has led to exponential increase in publications, news articles, and reports (Casillas et al., 2023). Significant knowledge gap exists in standardized analytical methods for the identification and quantification of MNPs from real world environmental samples. Here, we report comprehensive datasets utilizing thermogravimetric analyzer (TGA) coupled to a Fourier transformed infrared spectrometer (FTIR) and a gas chromatography/mass spectrometer (GC/MS) with corresponding Raman spectral data for the most common polymers documented to be present in the environment (35 plastics of 12 polymer types), to serve as a base line reference for the identification and quantitation of MNPs. Various parameters for TGA-FTIR-GC/MS data acquisition were optimized. Commercial consumer plastic product compositions were identified using this analytical database. Case studies to showcase the utility of the method for polymer mixtures analysis is included. This dataset would serve towards the development of a collaborative, global, comprehensive, and curated public database for the identification of various MNPs and mixtures.

Radiation impacts on toxicity of cobalt-chromium (CoCr) implant debris.

Particulate and soluble debris are generated by mechanical and non-mechanical degradation of implanted medical devices. Debris containing cobalt and chromium (CoCr) is known to cause adverse biological reactions. Implant-related complications are often diagnosed using radiography, which results in more frequent patient exposure to ionizing radiation. The aim of this study was to evaluate the potential for increased toxicity due to combined radiation and CoCr exposure. This was investigated using a controlled in vitro model consisting of commercially available CoCr debris that was generated from components of hip replacements and human cell lines relevant to the joint environment: endothelial HMEC-1 and synovial SW982. Particle sizes and shapes were heterogenous. Cells tended to internalize smaller particles, as observed by electron microscopy. Indicators of toxicity were measured after short (24 h after radiation) or extended (12-14 d after radiation) exposure timelines. In the short-term, CoCr reduced cell viability, increased apoptosis, and increased oxidative stress. The effects of radiation were not apparent until the timeline was extended. CoCr and radiation reduced cell survival, with both additive and synergistic effects. Mechanisms for reduced survival included rapid cell death caused by CoCr and senescence caused by radiation. In conclusion, results showed combined toxicological effects of CoCr and radiation at the doses and timelines used for this in vitro model. These observations warrant further investigation using other experimental models to determine translational impact.

Nanoparticle size and surface charge determine effects of PAMAM dendrimers on human platelets in vitro.

Blood platelets are essential in maintaining hemostasis. Various materials can activate platelets and cause them to aggregate. Platelet aggregation in vitro is often used as a marker for materials' thrombogenic properties, and studying nanomaterial interaction with platelets is an important step toward understanding their hematocompatibility. Here we report evaluation of 12 formulations of PAMAM dendrimers varying in size and surface charge. Using a cell counter based method, light transmission aggregometry and scanning electron microscopy, we show that only large cationic dendrimers, but not anionic, neutral or small cationic dendrimers, induce aggregation of human platelets in plasma in vitro. The aggregation caused by large cationic dendrimers was proportional to the number of surface amines. The observed aggregation was not associated with membrane microparticle release, and was insensitive to a variety of chemical and biological inhibitors known to interfere with various pathways of platelet activation. Taken in context with previously reported studies, our data suggest that large cationic PAMAM dendrimers induce platelet aggregation through disruption of membrane integrity.

U.S. Federal Agency interests and key considerations for new approach methodologies for nanomaterials.

Engineered nanomaterials (ENMs) come in a wide array of shapes, sizes, surface coatings, and compositions, and often possess novel or enhanced properties compared to larger sized particles of the same elemental composition. To ensure the safe commercialization of products containing ENMs, it is important to thoroughly understand their potential risks. Given that ENMs can be created in an almost infinite number of variations, it is not feasible to conduct in vivo testing on each type of ENM. Instead, new approach methodologies (NAMs) such as in vitro or in chemico test methods may be needed, given their capacity for higher throughput testing, lower cost, and ability to provide information on toxicological mechanisms. However, the different behaviors of ENMs compared to dissolved chemicals may challenge safety testing of ENMs using NAMs. In this study, member agencies within the Interagency Coordinating Committee on the Validation of Alternative Methods were queried about what types of ENMs are of agency interest and whether there is agency-specific guidance for ENM toxicity testing. To support the ability of NAMs to provide robust results in ENM testing, two key issues in the usage of NAMs, namely dosimetry and interference/bias controls, are thoroughly discussed.

Biological assessment of triazine dendrimer: toxicological profiles, solution behavior, biodistribution, drug release and efficacy in a PEGylated, paclitaxel construct.

The physicochemical characteristics, in vitro properties, and in vivo toxicity and efficacy of a third generation triazine dendrimer bearing approximately nine 2 kDa polyethylene glycol chains and twelve ester linked paclitaxel groups are reported. The hydrodynamic diameter of the neutral construct varies slightly with aqueous solvent ranging from 15.6 to 19.4 nm. Mass spectrometry and light scattering suggest radically different molecular weights with the former approximately 40 kDa mass consistent with expectation, and the latter 400 kDa mass consistent with a decameric structure and the observed hydrodynamic radii. HPLC can be used to assess purity as well as paclitaxel release, which is insignificant in organic solvents or aqueous solutions at neutral and low pH. Paclitaxel release occurs in vitro in human, rat, and mouse plasma and is nonlinear, ranging from 7 to 20% cumulative release over a 48 h incubation period. The construct is 2-3 orders of magnitude less toxic than Taxol by weight in human hepatocarcinoma (Hep G2), porcine renal proximal tubule (LLC-PK1), and human colon carcinoma (LS174T) cells, but shows similar cytotoxicity to Abraxane in LS174T cells. Both Taxol and the construct appear to induce caspase 3-dependent apoptosis. The construct shows a low level of endotoxin, is not hemolytic and does not induce platelet aggregation in vitro, but does appear to reduce collagen-induced platelet aggregation in vitro. Furthermore, the dendrimer formulation slightly activates the complement system in vitro due most likely to the presence of trace amounts (<1%) of free paclitaxel. An animal study provided insight into the maximum tolerated dose (MTD) wherein 10, 25, 50, and 100 mg of paclitaxel/kg of construct or Abraxane were administered once per week for three consecutive weeks to non tumor bearing athymic nude mice. The construct showed in vivo toxicity comparable to that of Abraxane. Both formulations were found to be nontoxic at the administered doses, and the dendrimer had an acute MTD greater than the highest dose administered. In a prostate tumor model (PC-3-h-luc), efficacy was observed over 70 days with an arrest of tumor growth and lack of luciferase activity observed in the twice treated cohort.

Chromatographic methods for the quantification of free and chelated gadolinium species in MRI contrast agent formulations.

Speciation measurements of gadolinium in liposomal MRI contrast agents (CAs) are complicated by the presence of emulsifiers, surfactants, and therapeutic agents in the formulations. The present paper describes two robust, hyphenated chromatography methods for the separation and quantification of gadolinium in nanoemulsion-based CA formulations. Three potential species of gadolinium, free gadolinium ion, gadolinium chelated by diethylenetriamine pentaacetic acid, and gadolinium chelated by 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid, were present in the CA formulations. The species were separated by reversed-phase chromatography (reversed phase high-performance liquid chromatography, RP-HPLC) or by high-pressure size-exclusion chromatography (HPSEC). For RP-HPLC, fluorescence detection and post-column online isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-MS) were used to measure the amount of gadolinium in each species. Online ID-ICP-MS and species-specific isotope dilution (SID)-ICP-MS were used in combination with the HPSEC column. The results indicated that some inter-species conversions and degradation had occurred within the samples and that SID-ICP-MS should be used to provide the most reliable measurements of total and speciated gadolinium. However, fluorescence and online ID-ICP-MS might usefully be applied as qualitative, rapid screening procedures for the presence of free gadolinium ions.

Effect of titanium dioxide nanoparticles on DNA methylation in multiple human cell lines.

Nanoscale titanium dioxide (TiO2) is manufactured in wide scale, with a range of applications in consumer products. Significant toxicity of TiO2 nanoparticles has, however, been recognized, suggesting considerable risk to human health. To evaluate fully their toxicity, assessment of the epigenetic action of these nanoparticles is critical. However, only few studies are available examining capability of nanoparticles to alter epigenetic integrity. In the present study, the effect of TiO2 nanoparticles exposure on DNA methylation, a major epigenetic mechanism, was investigated in in vitro cellular model systems. A panel of cells relevant to portals of human exposure (Caco-2 (colorectal), HepG2 (liver), NL20 (lung), and A-431 (skin)) was exposed to TiO2 nanoparticles to assess effects on global methylation, gene-specific methylation, and expression levels of DNA methyltransferases, MBD2, and UHRF1. Global methylation was determined by enzyme-linked immunosorbent assay-based immunochemical analysis. Degree of promoter methylation across a defined panel of genes was evaluated using EpiTect Methyl II Signature PCR System Array technology. Expression of DNMT1, DNMT3a, DNMT3b, MBD2, and URHF1 was quantified by qRT-PCR. Decrease in global DNA methylation in cell lines Caco-2, HepG2, and A-431 exposed to TiO2 nanoparticles was shown. Across four cell lines, eight genes (CDKN1A, DNAJC15, GADD45A, GDF15, INSIG1, SCARA3, TP53, and BNIP3) were identified in which promotors were methylated after exposure. Altered expression of these genes is associated with disease etiology. The results also revealed aberrant expression of epigenetic regulatory genes involved in DNA methylation (DNMT1, DNMT3a, DNMT3b, MBD2, and UHRF1) in TiO2 exposed cells, which was cell type dependent. Findings from this study clearly demonstrate the impact of TiO2 nanoparticles exposure on DNA methylation in multiple cell types, supporting potential involvement of this epigenetic mechanism in the toxicity of TiO2 nanoparticles. Hence for complete assessment of potential risk from nanoparticle exposure, epigenetic studies are critical.

Interaction of colloidal gold nanoparticles with human blood: effects on particle size and analysis of plasma protein binding profiles.

Nanoparticle size and plasma binding profile contribute to a particle's longevity in the bloodstream, which can have important consequences for therapeutic efficacy. In this study an approximate doubling in nanoparticle hydrodynamic size was observed upon in vitro incubation of 30- and 50-nm colloidal gold in human plasma. Plasma proteins that bind the surface of citrate-stabilized gold colloids have been identified. Effects of protein binding on the nanoparticle hydrodynamic size, elements of coagulation, and the complement system have been investigated. The difference in size measurements obtained from dynamic light scattering, electron microscopy, and scanning probe microscopy are also discussed.

HER2-specific affibody-conjugated thermosensitive liposomes (Affisomes) for improved delivery of anticancer agents.

Thermosensitive liposomes are attractive vehicles for the delivery and release of drugs to tumors. To improvethe targeting efficacy for breast cancer treatment, an 8.3-kDa HER2-specific Affibody molecule (Z(HER2:342)-Cys) was conjugated to the surface of liposomes. The effects of this modification on physical characteristics and stability of the resulting nanoparticles denoted as "Affisomes" were investigated. Thermosensitive small unilamellar vesicle (SUV) liposomes of (80-100 nm) a diameter consisting of dipalmitoyl phosphatidylcholine (DPPC, Tm 41 degrees C) as the matrix lipid and a maleimide-conjugated pegylated phospholipid (DSPE-MaL-PEG2000) were prepared by probe sonication. Fluorescent probes were incorporated into liposomes for biophysical and/or biochemical analysis and/or triggered-release assays. Affibody was conjugated to these liposomes via its C-terminal cysteine by incubation in the presence of a reducing agent (e.g., tributylphosphine) for 16-20 hours under an argon atmosphere. Lipid-conjugated affibody molecule was visible as an 11.3-kDa band on a 4-12% Bis/Tris gel under reducing conditions. Affibody conjugation yields were approximately 70% at a protein-lipid ratio of 20 microg/mg, with an average number of 200 affibody molecules per Affisome. Affibody conjugation to thermosensitive liposomes did not have any significant effect on the hydrodynamic size distribution of the liposomes. Thermosensitivity of Affisomes was determined by monitoring the release of entrapped calcein (a water-soluble fluorescent probe, lambdaex/em 490/515 nm) as a function of temperature. Calcein was released from Affisomes (thermosensitive liposomes with affibody-Targeted SUV) as well as nontargeted SUV (thermosensitive liposomes without affibody) in a temperature-dependent manner, with optimal leakage (90-100%) at 41 degrees C. In contrast, liposomes prepared from Egg phosphatidyl choline (Egg PC, Tm approximately 0 degrees C) under similar conditions released only 5-10% calcein at 41 degrees C. Affisomes, when stored at room temperature, retained > 90% entrapped calcein up to 7 days. Moreover, incubation of liposomes in phosphate-buffered saline, supplemented with 10% heat-inactivated serum (fetal bovine serum) did not result in a destabilization of liposomes. Therefore, Affisomes present promising, novel drug-delivery candidates for breast cancer targeting.

Comparative Evaluation of U.S. Brand and Generic Intravenous Sodium Ferric Gluconate Complex in Sucrose Injection: Physicochemical Characterization.

The objective of this study was to evaluate physicochemical equivalence between brand (i.e., Ferrlecit) and generic sodium ferric gluconate (SFG) in sucrose injection by conducting a series of comparative in vitro characterizations using advanced analytical techniques. The elemental iron and carbon content, thermal properties, viscosity, particle size, zeta potential, sedimentation coefficient, and molecular weight were determined. There was no noticeable difference between brand and generic SFG in sucrose injection for the above physical parameters evaluated, except for the sedimentation coefficient determined by sedimentation velocity analytical ultracentrifugation (SV-AUC) and molecular weight by asymmetric field flow fractionation-multi-angle light scattering (AFFF-MALS). In addition, brand and generic SFG complex products showed comparable molecular weight distributions when determined by gel permeation chromatography (GPC). The observed minor differences between brand and generic SFG, such as sedimentation coefficient, do not impact their biological activities in separate studies of in vitro cellular uptake and rat biodistribution. Coupled with the ongoing clinical study comparing the labile iron level in healthy volunteers, the FDA-funded post-market studies intended to illustrate comprehensive surveillance efforts ensuring safety and efficacy profiles of generic SFG complex in sucrose injection, and also to shed new light on the approval standards on generic parenteral iron colloidal products.

Investigating the susceptibility of mice to a bacterial challenge after intravenous exposure to durable nanoparticles.

AIM: The goal of this study was to determine whether bacterial clearance in a rodent model would be impaired upon exposure to gold, silver or silica nanoparticles (NPs). MATERIALS & METHODS: Mice received weekly injections of NPs followed by a challenge of Listeria monocytogenes (LM). On days 3 and 10 after LM injections, the animals were sacrificed and their tissues were collected for elemental analysis, electron microscopy and LM count determination. RESULTS: The untreated and NP-treated animals cleared LM at the same rate suggesting that bioaccumulation of NPs did not increase the animals' susceptibility to bacterial infection. CONCLUSION: The data from this study indicate that the bioaccumulation of NPs does not significantly affect the ability to react to a bacterial challenge.

Targeted drug delivery with dendrimers: comparison of the release kinetics of covalently conjugated drug and non-covalent drug inclusion complex.

Dendrimers have unique characteristics including monodispersity and modifiable surface functionality, along with highly defined size and structure. This makes these polymers attractive candidates as carriers in drug delivery applications. Drug delivery can be achieved by coupling a drug to polymer through one of two approaches. Hydrophobic drugs can be complexed within the hydrophobic dendrimer interior to make them water-soluble or drugs can be covalently coupled onto the surface of the dendrimer. Using both methods we compared the efficacy of generation 5 PAMAM dendrimers in the targeted drug delivery of methotrexate coupled to the polymer. The amine-terminated dendrimers bind to negatively charged membranes of cells in a non-specific manner and can cause toxicity in vitro and in vivo. To reduce toxicity and to increase aqueous solubility, modifications were made to the surface hydroxyl groups of the dendrimers. For targeted drug delivery, the dendrimer was modified to have a neutral terminal functionality for use with surface-conjugated folic acid as the targeting agent. The complexation of methotrexate within a dendrimer changes the water insoluble drug into a stable and readily water-soluble compound. When this dendrimer complexed drug, however, was placed in a solution of phosphate buffered saline, the methotrexate was immediately released and displayed diffusion characteristics identical to free methotrexate. Covalently coupled methotrexate dendrimer conjugates were stable under identical conditions in water and buffered saline. Cytotoxicity tests showed that methotrexate as the dendrimer inclusion complex had an activity identical to the free drug in vitro. In contrast, folic acid targeted dendrimer with covalently conjugated methotrexate specifically killed receptor-expressing cells by intracellular delivery of the drug through receptor-mediated endocytosis. This study demonstrates that while drug as a dendrimer inclusion complex is readily released and active in vitro, covalently conjugated drug to dendrimer is better suited for specifically targeted drug delivery.

Dendritic polymer macromolecular carriers for drug delivery.

Dendrimers are synthetic, highly branched, mono-disperse macromolecules of nanometer dimensions. Started in the mid-1980s, the research investigations into the synthetic methodology, physical and chemical properties of these macromolecules are increasing exponentially with growing interest in this field. Potential applications for dendrimers are now forthcoming. Properties associated with these dendrimers such as uniform size, water solubility, modifiable surface functionality and available internal cavities make them attractive for biological and drug-delivery applications.

Inhibition of phosphoinositol 3 kinase contributes to nanoparticle-mediated exaggeration of endotoxin-induced leukocyte procoagulant activity.

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.

Assessing the barriers to image-guided drug delivery.

Imaging has become a cornerstone for medical diagnosis and the guidance of patient management. A new field called image-guided drug delivery (IGDD) now combines the vast potential of the radiological sciences with the delivery of treatment and promises to fulfill the vision of personalized medicine. Whether imaging is used to deliver focused energy to drug-laden particles for enhanced, local drug release around tumors, or it is invoked in the context of nanoparticle-based agents to quantify distinctive biomarkers that could risk stratify patients for improved targeted drug delivery efficiency, the overarching goal of IGDD is to use imaging to maximize effective therapy in diseased tissues and to minimize systemic drug exposure in order to reduce toxicities. Over the last several years, innumerable reports and reviews covering the gamut of IGDD technologies have been published, but inadequate attention has been directed toward identifying and addressing the barriers limiting clinical translation. In this consensus opinion, the opportunities and challenges impacting the clinical realization of IGDD-based personalized medicine were discussed as a panel and recommendations were proffered to accelerate the field forward.