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.

Evaluating the potential of gold, silver, and silica nanoparticles to saturate mononuclear phagocytic system tissues under repeat dosing conditions.

BACKGROUND: As nanoparticles (NPs) become more prevalent in the pharmaceutical industry, questions have arisen from both industry and regulatory stakeholders about the long term effects of these materials. This study was designed to evaluate whether gold (10 nm), silver (50 nm), or silica (10 nm) nanoparticles administered intravenously to mice for up to 8 weeks at doses known to be sub-toxic (non-toxic at single acute or repeat dosing levels) and clinically relevant could produce significant bioaccumulation in liver and spleen macrophages. RESULTS: Repeated dosing with gold, silver, and silica nanoparticles did not saturate bioaccumulation in liver or spleen macrophages. While no toxicity was observed with gold and silver nanoparticles throughout the 8 week experiment, some effects including histopathological and serum chemistry changes were observed with silica nanoparticles starting at week 3. No major changes in the splenocyte population were observed during the study for any of the nanoparticles tested. CONCLUSIONS: The clinical impact of these changes is unclear but suggests that the mononuclear phagocytic system is able to handle repeated doses of nanoparticles.

How Has CDER Prepared for the Nano Revolution? A Review of Risk Assessment, Regulatory Research, and Guidance Activities.

The Nanotechnology Risk Assessment Working Group in the Center for Drug Evaluation and Research (CDER) within the United States Food and Drug Administration (FDA) was established to assess the potential impact of nanotechnology on drug products. One of the working group's major initiatives has been to conduct a comprehensive risk management exercise regarding the potential impact of nanomaterial pharmaceutical ingredients and excipients on drug product quality, safety, and efficacy. This exercise concluded that current review practices and regulatory guidance are capable of detecting and managing the potential risks to quality, safety, and efficacy when a drug product incorporates a nanomaterial. However, three risk management areas were identified for continued focus during the review of drug products containing nanomaterials: (1) the understanding of how to perform the characterization of nanomaterial properties and the analytical methods used for this characterization, (2) the adequacy of in vitro tests to evaluate drug product performance for drug products containing nanomaterials, and (3) the understanding of properties arising from nanomaterials that may result in different toxicity and biodistribution profiles for drug products containing nanomaterials. CDER continues to actively track the incorporation of nanomaterials in drug products and the methodologies used to characterize them, in order to continuously improve the readiness of our science- and risk-based review approaches. In parallel to the risk management exercise, CDER has also been supporting regulatory research in the area of nanotechnology, specifically focused on characterization, safety, and equivalence (between reference and new product) considerations. This article provides a comprehensive summary of regulatory and research efforts supported by CDER in the area of drug products containing nanomaterials and other activities supporting the development of this emerging technology.

The evolving landscape of drug products containing nanomaterials in the United States.

The Center for Drug Evaluation and Research (CDER) within the US Food and Drug Administration (FDA) is tracking the use of nanotechnology in drug products by building and interrogating a technical profile of products containing nanomaterials submitted to CDER. In this Analysis, data from more than 350 products show an increase in the submissions of drug products containing nanomaterials over the last two decades. Of these, 65% are investigational new drugs, 17% are new drug applications and 18% are abbreviated new drug applications, with the largest class of products being liposomal formulations intended for cancer treatments. Approximately 80% of products have average particle sizes of 300 nm or lower. This analysis identifies several trends in the development of drug products containing nanomaterials, including the relative rate of approvals of these products, and provides a comprehensive overview on the landscape of nanotechnology application in medicine.

Development Considerations for Nanocrystal Drug Products.

Nanocrystal technology has emerged as a valuable tool for facilitating the delivery of poorly water-soluble active pharmaceutical ingredients (APIs) and enhancing API bioavailability. To date, the US Food and Drug Administration (FDA) has received over 80 applications for drug products containing nanocrystals. These products can be delivered by different routes of administration and are used in a variety of therapeutic areas. To aid in identifying key developmental considerations for these products, a retrospective analysis was performed on the submissions received by the FDA to date. Over 60% of the submissions were for the oral route of administration. Based on the Biopharmaceutics Classification System (BCS), most nanocrystal drugs submitted to the FDA are class II compounds that possess low aqueous solubility and high intestinal permeability. Impact of food on drug bioavailability was reduced for most nanocrystal formulations as compared with their micronized counterparts. For all routes of administration, dose proportionality was observed for some, but not all, nanocrystal products. Particular emphasis in the development of nanocrystal products was placed on the in-process tests and controls at critical manufacturing steps (such as milling process), mitigation and control of process-related impurities, and the stability of APIs or polymorphic form (s) during manufacturing and upon storage. This emphasis resulted in identifying challenges to the development of these products including accurate determination of particle size (distribution) of drug substance and/or nanocrystal colloidal dispersion, identification of polymorphic form (s), and establishment of drug substance/product specifications.

Liposomal Drug Product Development and Quality: Current US Experience and Perspective.

Research in the area of liposomes has grown substantially in the past few decades. Liposomes are lipid bilayer structures that can incorporate drug substances to modify the drug's pharmacokinetic profile thereby improving drug delivery. The agency has received over 400 liposomal drug product submissions (excluding combination therapies), and there are currently eight approved liposomal drug products on the US market. In order to identify the pain points in development and manufacturing of liposomal drug products, a retrospective analysis was performed from a quality perspective on submissions for new and generic liposomal drug products. General analysis on liposomal drug product submissions was also performed. Results indicated that 96% of the submissions were Investigational New Drug (IND) applications, 3% were New Drug Applications (NDAs), and the remaining 1% was Abbreviated New Drug Applications (ANDAs). Doxorubicin hydrochloride was the most commonly used drug substance incorporated into the liposomes (31%). The majority of the liposomal products were administered via intravenous route (84%) with cancer (various types) being the most common indication (63%). From a quality perspective, major challenges during the development of liposomal drug products included identification and (appropriate) characterization of critical quality attributes of liposomal drug products and suitable control strategies during product development. By focusing on these areas, a faster and more efficient development of liposomal drug products may be achieved. Additionally, in this way, the drug review process for such products can be streamlined.

Effects of iron oxide nanoparticles on biological responses and MR imaging properties in human mammary healthy and breast cancer epithelial cells.

Superparamagnetic iron oxide nanoparticles (SPIONs, diameters >50 nm) have received great attention due to their promising use as magnetic resonance imaging (MRI) contrast agents. In this study, we evaluated the cellular uptake and biological responses in vitro of ultrasmall SPIONs (USPIONs, diameters < 50 nm). We compared the cellular responses between breast epithelia isolated from healthy and breast cancer donors after exposure to carboxy-terminated USPIONs (10 and 30 nm PEG-coated, 10 and 30 nm non-PEG-coated). The particles were characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS) and gel electrophoresis. Cellular interactions with USPIONs were assessed by confocal microscopy and TEM. Cellular uptake of USPIONs was quantified using ICP-MS. Cell viability was measured by MTT and neutral red uptake assays. T2* weighted MRI scans were performed using a 7T scanner. Results demonstrated that cell association/internalization of USPIONs was size- and surface coating-dependent (PEG vs. non-PEG), and higher cellular uptake of 10 and 30 nm non-coated particles was observed in both cell types compared with PEG-coated particles. Cell uptake for 10 and 30 nm non-coated particles was higher in cancer cells from two of three tested donors compared to healthy cells from three donors. There was no significant cytotoxicity observed for all tested particles. Significantly enhanced MRI contrast was observed following exposure to 10 and 30 nm non-coated particles compared to PEG-coated particles in both cell types. In comparison, cancer cells showed more enhanced MRI signals when compared to normal cells. The data indicate that cell responses following exposure to USPIONs are dependent on particle properties. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1032-1042, 2016.

Product quality for nanomaterials: current U.S. experience and perspective.

In recent years, there has been an increased focus on developing novel drug delivery systems and targeted therapies through the use of nanotechnology and nanomaterials. Such focus is translating to an increasing number of investigational new drug (IND) applications, new drug applications (NDAs), and abbreviated new drug applications (ANDAs) for drug products containing nanomaterials to the United States Food and Drug Administration (FDA). Although subject to the same rigorous regulatory standards and regulatory pathways as any drug product, unique properties that arise from the small size, large surface area, and polydispersity of nanomaterials may lead to additional scientific considerations when following current FDA guidelines and practices for drug evaluation. This review article will discuss these scientific considerations based on the experience with FDA-approved drug products containing nanomaterials.

Effect of silica and gold nanoparticles on macrophage proliferation, activation markers, cytokine production, and phagocytosis in vitro.

The accumulation of durable nanoparticles (NPs) in macrophages following systemic administration is well described. The ultimate biological impact of this accumulation on macrophage function, however, is not fully understood. In this study, nontoxic doses of two durable NPs, SiO2 and Au, at particle sizes of ~10 nm and 300 nm were used to evaluate the effect of bioaccumulation on macrophage function in vitro using RAW 264.7 mouse macrophage-like cells as a model system. Cell proliferation, cell cycle, cytokine production, surface marker activation, and phagocytosis responses were evaluated through a panel of assays using flow cytometry and confocal microscopy. The most dramatic change in RAW 264.7 cell function was a reduction in phagocytosis as monitored by the uptake of Escherichia coli. Cells exposed to both 10 nm Au NPs and 10 nm SiO2 NPs showed ~50% decrease in phagocytosis, while the larger NPs caused a less dramatic reduction. In addition to modifying phagocytosis profiles, 10 nm SiO2 NPs caused changes in proliferation, cell cycle, and cell morphology. Au NPs had no effect on cell cycle, cytokine production, or surface markers and caused interference in phagocytosis in the form of quenching when the assay was performed via flow cytometry. Confocal microscopy analysis was used to minimize this interference and demonstrated that both sizes of Au NPs decreased the phagocytosis of E. coli. Overall, our results demonstrate that Au and SiO2 NP uptake by macrophages can influence macrophage phagocytosis in vitro without altering surface markers and cytokine production in vitro. While the biological impact of these findings remains unclear, our results indicate that bioaccumulation of durable NPs within the macrophages may lead to a suppression of bacterial uptake and possibly impair bactericidal activity.

Evaluating the effect of assay preparation on the uptake of gold nanoparticles by RAW264.7 cells.

BACKGROUND: Cell culture conditions can greatly influence the results of nanoparticle (NP) uptake assays. In this study, 10 nm gold nanoparticles (AuNPs) and RAW 264.7 macrophages were used as a model system, while instrumental neutron activation analysis (NAA) was used as the elemental analysis technique to determine AuNP levels produced by the various culturing conditions. Static plate-based and insert-based culture conditions were compared with a dynamic suspension culture to evaluate the conditions' effect on the rate and extent of AuNP uptake. RESULTS: The results indicate that a dynamic culturing condition allows for the greatest NP uptake (approximately 3-5 times over the adherent conditions), whereas the plate-based assays have the initial highest rate of NP incorporation. CONCLUSIONS: These data highlight the importance of judiciously choosing the assay conditions prior to evaluating NP uptake.

Determination of nanoparticle surface coatings and nanoparticle purity using microscale thermogravimetric analysis.

The use of nanoparticles in some applications (i.e., nanomedical, nanofiltration, or nanoelectronic) requires small samples with well-known purities and composition. In addition, when nanoparticles are introduced into complex environments (e.g., biological fluids), the particles may become coated with matter, such as proteins or lipid layers. Many of today's analytical techniques are not able to address small-scale samples of nanoparticles to determine purity and the presence of surface coatings. Through the use of an elevated-temperature quartz crystal microbalance (QCM) method we call microscale thermogravimetric analysis, or µ-TGA, the nanoparticle purity, as well as the presence of any surface coatings of nanomaterials, can be measured. Microscale thermogravimetric analysis is used to determine the presence and amount of surface-bound ligand coverage on gold nanoparticles and confirm the presence of a poly(ethylene glycol) coating on SiO2 nanoparticles. Results are compared to traditional analytical techniques to demonstrate reproducibility and validity of µ-TGA for determining the presence of nanoparticle surface coatings. Carbon nanotube samples are also analyzed and compared to conventional TGA. The results demonstrate µ-TGA is a valid method for quantitative determination of the coatings on nanoparticles, and in some cases, can provide purity and compositional data of the nanoparticles themselves.

Nanosilver suppresses growth and induces oxidative damage to DNA in Caenorhabditis elegans.

Studies on the effects of nanomaterial exposure in mammals are limited, and new methods for rapid risk assessment of nanomaterials are urgently required. The utility of Caenorhabditis elegans cultured in axenic liquid media was evaluated as an alternative in vivo model for the purpose of screening nanomaterials for toxic effects. Spherical silver nanoparticles of 10 nm diameter (10nmAg) were used as a test material, and ionic silver from silver acetate as a positive control. Silver uptake and localization, larval growth, morphology and DNA damage were utilized as endpoints for toxicity evaluation. Confocal reflection analysis indicated that 10nmAg localized to the lumen and tissues of the digestive tract of C. elegans. 10nmAg at 10 µg ml(-1) reduced the growth of C. elegans larvae, and induced oxidative damage to DNA as measured by 8-OH guanine levels. Consistent with previously published studies using mammalian models, ionic silver suppressed growth in C. elegans larvae to a greater extent than 10nmAg. Our data suggest that medium-throughput growth screening and DNA damage analysis along with morphology assessments in C. elegans could together provide powerful tools for rapid toxicity screening of nanomaterials.

CDER risk assessment exercise to evaluate potential risks from the use of nanomaterials in drug products.

The Nanotechnology Risk Assessment Working Group in the Center for Drug Evaluation and Research (CDER) within the United States Food and Drug Administration was established to assess the possible impact of nanotechnology on drug products. The group is in the process of performing risk assessment and management exercises. The task of the working group is to identify areas where CDER may need to optimize its review practices and to develop standards to ensure review consistency for drug applications that may involve the application of nanotechnology. The working group already performed risk management exercises evaluating the potential risks from administering nanomaterial active pharmaceutical ingredients (API) or nanomaterial excipients by various routes of administration. This publication outlines the risk assessment and management process used by the working group, using nanomaterial API by the oral route of administration as an example.

Selective melamine detection in multiple sample matrices with a portable Raman instrument using surface enhanced Raman spectroscopy-active gold nanoparticles.

Melamine adulteration of food and pharmaceutical products is a major concern and there is a growing need to protect the public from exposure to contaminated or adulterated products. One approach to reduce this threat is to develop a portable method for on-site rapid testing. We describe a universal and selective method for the detection of melamine in a variety of solid matrices at the 100-200 µg L(-1) level by surface enhanced Raman spectroscopy (SERS) with gold nanoparticles. With minimal sample preparation and the use of a portable Raman spectrometer, this work will lead to field-based screening for melamine adulteration. Citrate coated gold nanoparticles (Au NPs) were investigated for both colorimetric and Raman-based responses. Several non-hazardous solvents were evaluated in order to develop a melamine extraction procedure safe for field applications. Au NP agglomerates formed by the addition of isopropanol (IPA) prior to sample introduction enhanced the Raman signal for melamine and eliminated matrix interference for substrate formation. The melamine Raman signal resulted in a 10(5) enhancement through the use of Au NP agglomerates. To our knowledge, we have developed the first portable SERS method using Au NPs to selectively screen for the presence of melamine adulteration in a variety of food and pharmaceutical matrices, including milk powder, infant formula, lactose, povidone, whey protein, wheat bran and wheat gluten.

Tumor necrosis factor interaction with gold nanoparticles.

We report on a systematic investigation of molecular conjugation of tumor necrosis factor-α (TNF) protein onto gold nanoparticles (AuNPs) and the subsequent binding behavior to its antibody (anti-TNF). We employ a combination of physical and spectroscopic characterization methods, including electrospray-differential mobility analysis, dynamic light scattering, polyacrylamide gel electrophoresis, attenuated total reflectance-Fourier transform infrared spectroscopy, fluorescence assay, and enzyme-linked immunosorbent assay. The native TNF used in this study exists in the active homotrimer configuration prior to conjugation. After binding to AuNPs, the maximum surface density of TNF is (0.09 ± 0.02) nm(-2) with a binding constant of 3 × 10(6) (mol L(-1))(-1). Dodecyl sulfate ions induce desorption of monomeric TNF from the AuNP surface, indicating a relatively weak intermolecular binding within the AuNP-bound TNF trimers. Anti-TNF binds to both TNF-conjugated and citrate-stabilized AuNPs, showing that non-specific binding is significant. Based on the number of anti-TNF molecules adsorbed, a substantially higher binding affinity was observed for the TNF-conjugated surface. The inclusion of thiolated polyethylene glycol (SH-PEG) on the AuNPs inhibits the binding of anti-TNF, and the amount of inhibition is related to the number ratio of surface bound SH-PEG to TNF and the way in which the ligands are introduced. This study highlights the challenges in quantitatively characterizing complex hybrid nanoscale conjugates, and provides insight on TNF-AuNP formation and activity.

Tissue and cellular distribution of gold nanoparticles varies based on aggregation/agglomeration status.

AIM: The ability of nanoparticles to form larger superstructures of aggregates and agglomerates has been extensively noted in the literature. The in vivo biological impact of these structures, however, has not been assessed. This knowledge gap is especially critical in the safety assessment of nanoparticles to be used for therapeutic purposes. METHOD/RESULTS: Here we show that when administered intravenously into a mouse model, gold nanoparticle superstructures of reversible agglomerates and irreversible aggregates demonstrate significant differences in organ and cellular distribution compared with the primary particle building blocks. In addition, different structures produced different blood serum chemistry data. CONCLUSION: These findings raise the possibility for different mechanisms of toxicity between the structures. Such a possibility necessitates complete characterization and stability assessment of nanomaterials prior to their in vivo administration.

Current perspectives on the US FDA regulatory framework for intelligent drug-delivery systems.

The US FDA is the US agency responsible for regulating intelligent drug-delivery systems (IDDS). IDDS can be classified as a device, drug, biologic or combination product. In this perspective, the current regulatory framework for IDDS and future perspectives on how the field is expected to evolve from a regulatory standpoint is discussed.

Adsorption and conformation of serum albumin protein on gold nanoparticles investigated using dimensional measurements and in situ spectroscopic methods.

The adsorption and conformation of bovine serum albumin (BSA) on gold nanoparticles (AuNPs) were interrogated both qualitatively and quantitatively via complementary physicochemical characterization methods. Dynamic light scattering (DLS), asymmetric-flow field flow fractionation (AFFF), fluorescence spectrometry, and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy were combined to characterize BSA-AuNP conjugates under fluid conditions, while conjugates in the aerosol state were characterized by electrospray-differential mobility analysis (ES-DMA). The presence of unbound BSA molecules interferes with DLS analysis of the conjugates, particularly as the AuNP size decreases (i.e., below 30 nm in diameter). Under conditions where the γ value is high, where γ is defined as the ratio of scattering intensity by AuNPs to the scattering intensity by unbound BSA, DLS size results are consistent with results obtained after fractionation by AFFF. Additionally, the AuNP hydrodynamic size exhibits a greater proportional increase due to BSA conjugation at pH values below 2.5 compared with less acidic pH values (3.4-7.3), corresponding with the reversibly denatured (E or F form) conformation of BSA below pH 2.5. Over the pH range from 3.4 to 7.3, the hydrodynamic size of the conjugate is nearly constant, suggesting conformational stability over this range. Because of the difference in the measurement environment, a larger increase of AuNP size is observed following BSA conjugation when measured in the wet state (i.e., by DLS and AFFF) compared to the dry state (by ES-DMA). Molecular surface density for BSA is estimated based on ES-DMA and fluorescence measurements. Results from the two techniques are consistent and similar, but slightly higher for ES-DMA, with an average adsorbate density of 0.015 nm(-2). Moreover, from the change of particle size, we determine the extent of adsorption for BSA on AuNPs using DLS and ES-DMA at 21 °C, which show that increasing the concentration of BSA increases the measured change in AuNP size. Using ES-DMA, we observe that the BSA surface density reaches 90% of saturation at a solution phase concentration between 10 and 30 µmol/L, which is roughly consistent with fluorescence and ATR-FTIR results. The equilibrium binding constant for BSA on AuNPs is calculated by applying the Langmuir equation, with resulting values ranging from 0.51 × 10(6) to 1.65 × 10(6) L/mol, suggesting a strong affinity due to bonding between the single free exterior thiol on N-form BSA (associated with a cysteine residue) and the AuNP surface. Moreover, the adsorption interaction induces a conformational change in BSA secondary structure, resulting in less α-helix content and more open structures (ß-sheet, random, or expanded).

Gold nanoparticle trafficking of typically excluded compounds across the cell membrane in JB6 Cl 41-5a cells causes assay interference.

Nanoparticles (NP) often interfere with the mechanism and interpretation of high throughput in vitro toxicity assays. This interference may occur at any time during the assay and spans most NP systems. This study reports on a specific type of gold NP assay interference, where unmodified gold NPs were able to traffic certain assay molecules that contained primary amines across the cell membrane resulting in false positive results for toxicity assays. The enhanced assay molecule permeability was eliminated when the gold NP surface was both sterically and chemically blocked by polyethylene glycol (PEG). The results support the growing consensus that appropriate controls and assay validation should occur prior to interpretation of results of assays using NP.