Is this child suitable to be seen in primary care? Poor agreement between caregiver/family perception and definitions of a 'primary care-type' patient.

AIMS: To compare and evaluate the number of paediatric patients classified as 'suitable for primary care' using the Australian Institute of Health and Welfare (AIHW) method, the Australasian College for Emergency Medicine (ACEM) method, and parental judgement. METHODS: This was a prospective observational study enrolling parents/carers presenting with their children to two Victorian EDs in Victoria, Australia over a 1-week period. Trained research assistants were posted within both EDs and surveyed all eligible parents/carers whether they agreed with the statement 'I think a GP would be able to look after my child's current illness/injury'. Survey responses were linked to clinical outcomes and length of stay. Each presentation was classified as suitable for primary care using the AIHW method, the ACEM method and parental survey. Agreement between definitions was assessed using Cohen's kappa statistic. RESULTS: During the study (June 2016), 1069 patients presented to the two EDs; 677 patients were able to be classified under all three definitions (AIHW: 1069, ACEM: 991, survey: 677 patients). Only 80/677 (12%) patients met all three criteria. Agreement was slight between the parent survey and the ACEM method (K = 0.14, 95% confidence interval (CI) 0.06-0.21), and the parent survey and the AIHW method (K = 0.12, 95% CI 0.05-0.19). There was moderate agreement between the ACEM and AIHW methods (K = 0.45, 95% CI 0.39-0.51). CONCLUSIONS: There is very poor agreement on what defines a 'primary care-type' paediatric patient between the definitions used by government, professional bodies and caregivers.

Preclinical Evaluation of [155/161Tb]Tb-Crown-TATE-A Novel SPECT Imaging Theranostic Agent Targeting Neuroendocrine Tumours.

Terbium radioisotopes (149Tb, 152Tb, 155Tb, 161Tb) offer a unique class of radionuclides which encompass all four medicinally relevant nuclear decay modalities (α, ß+, γ, ß-/e-), and show high potential for the development of element-matched theranostic radiopharmaceuticals. The goal of this study was to design, synthesise, and evaluate the suitability of crown-TATE as a new peptide-conjugate for radiolabelling of [155Tb]Tb3+ and [161Tb]Tb3+, and to assess the imaging and pharmacokinetic properties of each radiotracer in tumour-bearing mice. [155Tb]Tb-crown-TATE and [161Tb]Tb-crown-TATE were prepared efficiently under mild conditions, and exhibited excellent stability in human serum (>99.5% RCP over 7 days). Longitudinal SPECT/CT images were acquired for 155Tb- and 161Tb- labelled crown-TATE in male NRG mice bearing AR42J tumours. The radiotracers, [155Tb]Tb-crown-TATE and [161Tb]Tb-crown-TATE, showed high tumour targeting (32.6 and 30.0 %ID/g, respectively) and minimal retention in non-target organs at 2.5 h post-administration. Biodistribution studies confirmed the SPECT/CT results, showing high tumour uptake (38.7 ± 8.0 %ID/g and 38.5 ± 3.5 %ID/g, respectively) and favourable tumour-to-background ratios. Blocking studies further confirmed SSTR2-specific tumour accumulation. Overall, these findings suggest that crown-TATE has great potential for element-matched molecular imaging and radionuclide therapy using 155Tb and 161Tb.

Zeta potential: a case study of cationic, anionic, and neutral liposomes.

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.

Anticoagulants Influence the Performance of In Vitro Assays Intended for Characterization of Nanotechnology-Based Formulations.

The preclinical safety assessment of novel nanotechnology-based drug products frequently relies on in vitro assays, especially during the early stages of product development, due to the limited quantities of nanomaterials available for such studies. The majority of immunological tests require donor blood. To enable such tests one has to prevent the blood from coagulating, which is usually achieved by the addition of an anticoagulant into blood collection tubes. Heparin, ethylene diamine tetraacetic acid (EDTA), and citrate are the most commonly used anticoagulants. Novel anticoagulants such as hirudin are also available but are not broadly used. Despite the notion that certain anticoagulants may influence assay performance, a systematic comparison between traditional and novel anticoagulants in the in vitro assays intended for immunological characterization of nanotechnology-based formulations is currently not available. We compared hirudin-anticoagulated blood with its traditional counterparts in the standardized immunological assay cascade, and found that the type of anticoagulant did not influence the performance of the hemolysis assay. However, hirudin was more optimal for the complement activation and leukocyte proliferation assays, while traditional anticoagulants citrate and heparin were more appropriate for the coagulation and cytokine secretion assays. The results also suggest that traditional immunological controls such as lipopolysaccharide (LPS ) are not reliable for understanding the role of anticoagulant in the assay performance. We observed differences in the test results between hirudin and traditional anticoagulant-prepared blood for nanomaterials at the time when no such effects were seen with traditional controls. It is, therefore, important to recognize the advantages and limitations of each anticoagulant and consider individual nanoparticles on a case-by-case basis.

Quantitative analysis of PEG-functionalized colloidal gold nanoparticles using charged aerosol detection.

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.

Analyzing the influence of PEG molecular weight on the separation of PEGylated gold nanoparticles by asymmetric-flow field-flow fractionation.

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.

Induction of oxidative stress by Taxol® vehicle Cremophor-EL triggers production of interleukin-8 by peripheral blood mononuclear cells through the mechanism not requiring de novo synthesis of mRNA.

Understanding the ability of cytotoxic oncology drugs, and their carriers and formulation excipients, to induce pro-inflammatory responses is important for establishing safe and efficacious formulations. Literature data about cytokine response induction by the traditional formulation of paclitaxel, Taxol®, are controversial, and no data are available about the pro-inflammatory profile of the nano-albumin formulation of this drug, Abraxane®. Herein, we demonstrate and explain the difference in the cytokine induction profile between Taxol® and Abraxane®, and describe a novel mechanism of cytokine induction by a nanosized excipient, Cremophor EL, which is not unique to Taxol® and is commonly used in the pharmaceutical industry for delivery of a wide variety of small molecular drugs. FROM THE CLINICAL EDITOR: Advances in nanotechnology have enabled the production of many nano-formulation drugs. The cellular response to drugs has been reported to be different between traditional and nano-formulations. In this article, the authors investigated and compared cytokine response induction profiles between Taxol® and Abraxane®. The findings here provided further understanding to create drugs with better safety profiles.

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.

Strategic aspects for the commercialization of nanomedicines.

The projected growth of the nanomedicine market mirrors the increase in commercial interest and investment in the field. Yet, amidst this optimism, research efforts have often been geared towards developing innovative materials, and less on bringing these innovations to market. In this article, we present a strategic approach of 'commercialization by design' to overcome various challenges related to commercialization. This approach shifts the focus from materials-centric development to one driven by market demands, evaluating nanomedicines considering factors like reimbursement restrictions and unmet medical needs, and aiming to generate robust evidence for regulatory authorities, Health Technology Assessment bodies and Payers alike. We highlight the linkages from the market backwards to the preclinical phase, where nanomedicines are tailored to specific clinician-identified requirements. Transitioning from a technology 'push' model to one with market 'pull' will enhance the commercial success of nanomedicines.

Preclinical evaluation of MC1R targeting theranostic pair [155Tb]Tb-crown-αMSH and [161Tb]Tb-crown-αMSH.

BACKGROUND: Targeted radionuclide therapy is established as a highly effective strategy for the treatment of metastatic tumors; however, the co-development of suitable imaging companions to therapy remains significant challenge. Theranostic isotopes of terbium (149Tb, 152Tb, 155Tb, 161Tb) have the potential to provide chemically identical radionuclidic pairs, which collectively encompass all modes of nuclear decay relevant to nuclear medicine. Herein, we report the first radiochemistry and preclinical studies involving 155Tb- and 161Tb-labeled crown-αMSH, a small peptide-based bioconjugate suitable for targeting melanoma. METHODS: 155Tb was produced via proton induced spallation of Ta targets using the isotope separation and acceleration facility at TRIUMF with isotope separation on-line (ISAC/ISOL). The radiolabeling characteristics of crown-αMSH with 155Tb and/or 161Tb were evaluated by concentration-dependence radiolabeling studies, and radio-HPLC stability studies. LogD7.4 measurements were obtained for [161Tb]Tb-crown-αMSH. Competitive binding assays were undertaken to determine the inhibition constant for [natTb]Tb-crown-αMSH in B16-F10 cells. Pre-clinical biodistribution and SPECT/CT imaging studies of 155Tb and 161Tb labeled crown-αMSH were undertaken in male C57Bl/6 J mice bearing B16-F10 melanoma tumors to evaluate tumor specific uptake and imaging potential for each radionuclide. RESULTS: Quantitative radiolabeling of crown-αMSH with [155Tb]Tb3+ and [161Tb]Tb3+ was demonstrated under mild conditions (RT, 10 min) and low chelator concentrations; achieving high molar activities (23-29 MBq/nmol). Radio-HPLC studies showed [161Tb]Tb-crown-αMSH maintains excellent radiochemical purity in human serum, while gradual metabolic degradation is observed in mouse serum. Competitive binding assays showed the high affinity of [natTb]Tb-crown-αMSH toward MC1R. Two different methods for preparation of the [155Tb]Tb-crown-αMSH radiotracer were investigated and the impacts on the biodistribution profile in tumor bearing mice is compared. Preclinical in vivo studies of 155Tb- and 161Tb- labeled crown-αMSH were performed in parallel, in mice bearing B16-F10 tumors; where the biodistribution results showed similar tumor specific uptake (6.06-7.44 %IA/g at 2 h pi) and very low uptake in nontarget organs. These results were further corroborated through a series of single-photon emission computed tomography (SPECT) studies, with [155Tb]Tb-crown-αMSH and [161Tb]Tb-crown-αMSH showing comparable uptake profiles and excellent image contrast. CONCLUSIONS: Collectively, our studies highlight the promising characteristics of [155Tb]Tb-crown-αMSH and [161Tb]Tb-crown-αMSH as theranostic pair for nuclear imaging (155Tb) and radionuclide therapy (161Tb).

Polymeric curcumin nanoparticle pharmacokinetics and metabolism in bile duct cannulated rats.

The objective of this study was to compare the pharmacokinetics and metabolism of polymeric nanoparticle-encapsulated (nanocurcumin) and solvent-solubilized curcumin formulations in Sprague-Dawley (SD) rats. Nanocurcumin is currently under development for cancer therapy. Since free, unencapsulated curcumin is rapidly metabolized and excreted in rats, upon intravenous (i.v.) administration of nanocurcumin only nanoparticle-encapsulated curcumin can be detected in plasma samples. Hence, the second objective of this study was to utilize the metabolic instability of curcumin to assess in vivo drug release from nanocurcumin. Nanocurcumin and solvent-solubilized curcumin were administered at 10 mg curcumin/kg by jugular vein to bile duct-cannulated male SD rats (n = 5). Nanocurcumin increased the plasma Cmax of curcumin 1749 fold relative to the solvent-solubilized curcumin. Nanocurcumin also increased the relative abundance of curcumin and glucuronides in bile but did not dramatically alter urine and tissue metabolite profiles. The observed increase in biliary and urinary excretion of both curcumin and metabolites for the nanocurcumin formulation suggested a rapid "burst" release of curcumin. Although the burst release observed in this study is a limitation for targeted tumor delivery, nanocurcumin still exhibits major advantages over solvent-solubilized curcumin, as the nanoformulation does not result in the lung accumulation observed for the solvent-solubilized curcumin and increases overall systemic curcumin exposure. Additionally, the remaining encapsulated curcumin fraction following burst release is available for tumor delivery via the enhanced permeation and retention effect commonly observed for nanoparticle formulations.

Cyclotron production of 103Pd using a liquid target.

INTRODUCTION: In this work, we present the first feasibility study on the production of the medically important radionuclide 103Pd via the 103Rh(p,n)103Pd reaction by cyclotron irradiation of a liquid target. Using a liquid target removes the time consuming and complex dissolution process of rhodium post-irradiation due to its chemically inactive nature and thereby will improve the accessibility of this radioisotope. METHODS: Liquid targets made from Rh(NO3)3·×H2O salt dissolved in de-ionized water were irradiated using a 12 MeV beam at the TR13 cyclotron at TRIUMF, Vancouver. RESULTS: A maximum EOB activity of 1.03 ± 0.05 MBq was achieved with the tested conditions, sufficient for basic radiochemistry studies. An effective separation method using anion exchange chromatography is reported using 1 M HNO3 as an eluent for rhodium (90.1 ± 2.1 % recovery) and a 1:1 mixture of 0.5 M NH3 + NH4Cl palladium eluent (103.8 ± 2.3 % recovery). The solution showed good in-target pressure stability. However, the production efficiency decreased significantly with higher solution concentrations and irradiation lengths which puts into question the scaling potential of this method. CONCLUSION: This proof-of-concept study has demonstrated the potential for using liquid targets as complementary production method of 103Pd for research purposes. The liquid target route faces several scaling challenges but can nonetheless improve the availability of 103Pd and consequently aid in widening its utility for radiopharmaceuticals.

Detection, management, and prevention of diabetes-related foot disease in the Australian context.

Diabetes-related foot disease (DFD) is a widely feared complication among people who live with diabetes. In Australia and globally, rates of disability, cardio-vascular disease, lower extremity amputation, and mortality are significantly increased in patients with DFD. In order to understand and prevent these outcomes, we analyse the common pathogenetic processes of neuropathy, arterial disease, and infection. The review then summarises important management considerations through the interdisciplinary lens. Using Australian and international guidelines, we offer a stepwise, evidence-based practical approach to the care of patients with DFD.

Determination of distribution coefficients of mercury and gold on selected extraction chromatographic resins - towards an improved separation method of mercury-197 from proton-irradiated gold targets.

Radioisotope mercury-197g (197gHg, half-life: 64.14 h) along with its metastable isomer (197mHg, half-life: 23.8 h) are potential candidates for targeted Meitner-Auger electron therapy due to their suitable decay properties. Their production can be achieved via proton irradiation of a natural gold target, but the number of studies surrounding their separation from an irradiated gold target is limited. This study focuses on the determination of distribution coefficients (Kd) of gold (III) and mercury (II) on seven extraction chromatographic resins. Mercury Kd were measured by means of radiotracers and Inductively Coupled Plasma Mass Spectrometry (ICP_MS); values obtained from the two methods were generally in good agreement. These results can provide insight on Hg and Au chemistry and aid in the design of improved separation system(s).

Optimized production, purification, and radiolabeling of the 203Pb/212Pb theranostic pair for nuclear medicine.

TRIUMF is one of the only laboratories in the world able to produce both lead-203 (203Pb, t1/2 = 51.9 h) and 212Pb (t1/2 = 10.6 h) onsite via its 13 and 500 MeV cyclotrons, respectively. Together, 203Pb and 212Pb form an element-equivalent theranostic pair that potentiate image-guided, personalized cancer treatment, using 203Pb as a single-photon emission computed tomography (SPECT) source, and 212Pb for targeted alpha therapy. In this study, improvements to 203Pb production were accomplished by manufacturing electroplated, silver-backed thallium (Tl) targets to improve target thermal stability, which allow for higher currents during irradiation. We implemented a novel, two-column purification method that employs selective Tl precipitation (203Pb only) alongside extraction and anion exchange chromatography to elute high specific activity and chemical purity 203/212Pb in a minimal volume of dilute acid, without the need for evaporation. Optimization of the purification method translated to improvements in radiolabeling yields and apparent molar activity of lead chelators TCMC (S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra(2-carbamoylmethyl)cyclododecane) and Crypt-OH, a derivative of a [2.2.2]-cryptand.

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.

A simple and automated method for 161Tb purification and ICP-MS analysis of 161Tb.

BACKGROUND: 161Tb is a radiolanthanide with the potential to replace 177Lu in targeted radionuclide therapy. 161Tb is produced via the neutron irradiation of [160Gd]Gd2O3 targets, and must be purified from 160Gd and the decay product 161Dy prior to use. Established purification methods require complex conditions or high-pressure ion chromatography (HPIC) which are inconvenient to introduce in a broad user community. This study aims to find a simpler small solid-phase extraction (SPE) column method for 161Tb purification that is more suitable for automation with commercially available systems like TRASIS. RESULTS: We first tested the distribution coefficients on TK211 and TK212 resins for the separation of Gd, Tb, and Dy, and subsequently developed a method to separate these metal ions, with an additional TK221 resin to concentrate the final product. A side-by-side comparison of the products purified using this new method with the HPIC method was undertaken, assessing the radionuclidic purity, chemical purity regarding Gd and Dy, and labeling efficiency with a standard chelate (DOTA) and a novel chelate (crown). The two methods have comparable radionuclidic purity and labeling efficiency. The small SPE column method reduced Gd content to nanogram level, although still higher than the HPIC method. An ICP-MS method to quantify 161Tb, 159Tb, 160Gd, and 161Dy was developed with the application of mass-shift by ammonia gas. Last, 161Tb produced from the small SPE column method was used to assess the biodistribution of [161Tb]Tb-crown-αMSH, and the results were comparable to the HPIC produced 161Tb. CONCLUSIONS: 161Tb was successfully purified by a semi-automated TRASIS system using a combination of TrisKem extraction resins. The resulting product performed well in radiolabelling and in vivo experiments. However, improvement can be made in the form of further reduction of 160Gd target material in the final product. An ICP-MS method to analyze the radioactive product was developed. Combined with gamma spectroscopy, this method allows the purity of 161Tb being assessed before the decay of the product, providing a useful tool for quality control.

A pragmatic regulatory approach for complex generics through the U.S. FDA 505(j) or 505(b)(2) approval pathways.

The diverse nature of complex drug products poses challenges for the development of regulatory guidelines for generic versions. While complexity is not new in medicines, the technical capacity to measure and analyze data has increased. This requires a determination of which measurements and studies are relevant to demonstrate therapeutic equivalence. This paper describes the views of the NBCD Working Group and provides pragmatic solutions for approving complex generics by making best use of existing U.S. Food and Drug Administration's abbreviated approval pathways 505(j) and 505(b)(2). We argue that decisions on the appropriateness of submitting a 505(j) or 505(b)(2) application can build on the FDA's complex drug product classification as well as the FDA's much applauded guidance document for determining whether to submit an ANDA or a 505(b)(2) application. We hope that this paper contributes to the discussions to increase the clarity of regulatory approaches for complex generics, as well as the predictability for complex generic drug developers, to facilitate access to much-needed complex generics and to promote the sustainability of the healthcare system.

Fullerenol cytotoxicity in kidney cells is associated with cytoskeleton disruption, autophagic vacuole accumulation, and mitochondrial dysfunction.

Water soluble fullerenes, such as the hydroxylated fullerene, fullerenol (C60OHx), are currently under development for diagnostic and therapeutic biomedical applications in the field of nanotechnology. These molecules have been shown to undergo urinary clearance, yet there is limited data available on their renal biocompatibility. Here we examine the biological responses of renal proximal tubule cells (LLC-PK1) exposed to fullerenol. Fullerenol was found to be cytotoxic in the millimolar range, with viability assessed by the sulforhodamine B and trypan blue assays. Fullerenol-induced cell death was associated with cytoskeleton disruption and autophagic vacuole accumulation. Interaction with the autophagy pathway was evaluated in vitro by Lysotracker Red dye uptake, LC3-II marker expression and TEM. Fullerenol treatment also resulted in coincident loss of cellular mitochondrial membrane potential and ATP depletion, as measured by the Mitotracker Red dye and the luciferin-luciferase assays, respectively. Fullerenol-induced ATP depletion and loss of mitochondrial potential were partially ameliorated by co-treatment with the autophagy inhibitor, 3-methyladenine. In vitro fullerenol treatment did not result in appreciable oxidative stress, as measured by lipid peroxide and glutathione content. Based on these data, it is hypothesized that cytoskeleton disruption may be an initiating event in fullerenol cytotoxicity, leading to subsequent autophagy dysfunction and loss of mitochondrial capacity. As nanoparticle-induced cytoskeleton disruption, autophagic vacuole accumulation and mitochondrial dysfunction are commonly reported in the literature, the proposed mechanism may be relevant for a variety of nanomaterials.

Energy dispersive X-ray analysis of titanium dioxide nanoparticle distribution after intravenous and subcutaneous injection in mice.

In an effort to understand the disposition and toxicokinetics of nanoscale materials, we used EDS (energy dispersive X-ray spectroscopy) to detect and map the distribution of titanium dioxide (TiO2) in tissue sections from mice following either subcutaneous (s.c.) or intravenous (i.v.) injection. TiO2 nanoparticles were administered at a dose of 560 mg/kg (i.v.) or 5600 mg/kg (s.c.) to Balb/c female mice on two consecutive days. Tissues (liver, kidney, lung, heart, spleen, and brain) were examined by light microscopy, TEM (transmission electron microscopy), SEM (scanning electron microscopy), and EDS following necropsy one day after treatment. Particle agglomerates were detected by light microscopy in all tissues examined, EDS microanalysis was used to confirm that these tissues contained elemental titanium and oxygen. The TEM micrographs and EDS spectra of the aggregates were compared with in vitro measurements of TiO2 nanoparticle injection solution (i.e., in water). The nanoparticles were also characterized using dynamic light scattering in water, 10 mM NaCl, and phosphate buffered saline (PBS). In low ionic strength solvents (water and 10 mM NaCl), the TiO2 particles had average hydrodynamic diameters ranging from 114-122 nm. In PBS, however, the average diameter increases to 1-2 microm, likely due to aggregation analogous to that observed in tissue by TEM and EDS. This investigation demonstrates the suitability of energy dispersive X-ray spectroscopy (EDS) for detection of nanoparticle aggregates in tissues and shows that disposition of TiO2 nanoparticles depends on the route of administration (i.v. or s.c.).