Integration of native bone into orthopedic devices is a key factor in long-term implant success. The material-tissue interface is generally accepted to consist of a hydroxyapatite layer so bioactive materials that can spontaneously generate this hydroxyapatite layer after implantation may improve patient outcomes. Per the ISO 22317:2014 standard, "Implants for surgery - In vitro evaluation for apatite-forming ability of implant materials," bioactivity performance statements can be assessed by soaking the material in simulated body fluid (SBF) and evaluating the surface for the formation of a hydroxyapatite layer; however, variations in test methods may alter hydroxyapatite formation and result in false-positive assessments. The goal of this study was to identify the effect of SBF formulation on bioactivity assessment. Bioglass® (45S5 and S53P4) and non-bioactive Ti-6Al-4V were exposed to SBF formulations varying in calcium ion and phosphate concentrations as well as supporting ion concentrations. Scanning electron microscopy and X-ray powder diffraction evaluation of the resulting hydroxyapatite layers revealed that SBF enriched with double or quadruple the calcium and phosphate ion concentrations increased hydroxyapatite crystal size and quantity compared to the standard formulation and can induce hydroxyapatite crystallization on surfaces traditionally considered non-bioactive. Altering concentrations of other ions, for example, bicarbonate, changed hydroxyapatite induction time, quantity, and morphology. For studies evaluating the apatite-forming ability of a material to support bioactivity performance statements, test method parameters must be adequately described and controlled. It is unclear if apatite formation after exposure to any of the SBF formulations is representative of an in vivo biological response. The ISO 23317 standard test method should be further developed to provide additional guidance on apatite characterization and interpretation of the results.

Propofol is intravenously administered oil-in-water emulsion stabilized by egg lecithin phospholipids indicated for the induction and maintenance of general anesthesia or sedation. It is generally assumed to be structurally homogenous as characterized by commonly used dynamic light scattering technique and laser diffraction. However, the excessive amount of egg lecithin phospholipids added to the propofol formulation may, presumably, give rise to additional formation of lipid vesicles (i.e., vesicular structures consisting of a phospholipid bilayer). In this study, we investigate the use of high-resolution cryogenic transmission electron microscopy (cryo-TEM) in morphological characterization of four commercially available propofol drug products. The TEM result, for the first time, reveals that all propofol drug products contain lipid vesicles and oil droplet-lipid vesicle aggregated structures, in addition to oil droplets. Statistical analysis shows the size and ratio of the lipid vesicles varies across four different products. To evaluate the impact of such morphological differences on active pharmaceutical ingredient (API)'s distribution, we separate the lipid vesicle phase from other constituents via ultracentrifuge fractionation and determine the amount of propofol (2,6-diisopropylphenol) using high performance liquid chromatography (HPLC). The results indicate that a nearly negligible amount of API (i.e., NMT 0.25% of labeled content) is present in the lipid vesicles and is thus primarily distributed in the oil phase. As oil droplets are the primary drug carriers and their globule size are similar, the findings of various lipid vesicle composition and sizes among different propofol products do not affect their clinical outcomes.

Ultrasmall superparamagnetic iron oxide nanoparticles (USPION) possess reactive surfaces, are metabolized and exhibit unique magnetic properties. These properties are desirable for designing novel theranostic biomedical products; however, toxicity mechanisms of USPION are not completely elucidated. The goal of this study was to investigate cell interactions (uptake and cytotoxicity) of USPION using human coronary artery endothelial cells as a vascular cell model. Polyvinylpirrolidone-coated USPION were characterized: average diameter 17 nm (transmission electron microscopy [TEM]), average hydrodynamic diameter 44 nm (dynamic light scattering) and zeta potential -38.75 mV. Cells were exposed to 0 (control), 25, 50, 100 or 200 μg/mL USPION. Concentration- and time-dependent cytotoxicity were observed after 3-6 hours through 24 hours of exposure using Alamar Blue and Real-Time Cell Electronic Sensing assays. Cell uptake was evaluated by imaging using live-dead confocal microscopy, actin and nuclear fluorescent staining, and TEM. Phase-contrast, confocal microscopy, and TEM imaging showed significant USPION internalization as early as 3 hours after exposure to 25 μg/mL. TEM imaging demonstrated particle internalization in secondary lysosomes with perinuclear localization. Three orthogonal assays were conducted to assess apoptosis. TUNEL staining demonstrated a marked increase in fragmented DNA, a response pathognomonic of apoptosis, after a 4-hour exposure. Cells subjected to agarose gel electrophoresis exhibited degraded DNA 3 hours after exposure. Caspase-3/7 activity increased after a 3-hour exposure. USPION uptake resulted in cytotoxicity involving apoptosis and these results contribute to further mechanistic understanding of the USPION toxicity in vitro in cardiovascular endothelial cells.

Identifying the critical process parameters (CPPs) of a complex drug product manufacture and the associated impact on critical quality attributes (CQAs) is essential to the development and quality control of both new and generic drugs. AmBisome, a liposomal amphotericin B (AMB) macrolide antibiotic widely adopted as an important antifungal drug product, was used as a model complex drug product in the current study. This study investigated how multi-step production approaches and related manufacturing conditions may affect essential physico-chemical and toxicological properties of the final drug product. A key challenge in the manufacture and analysis of liposomal AMB was the drug substance's propensity to aggregate, with associated poor solubility in water and organic solvents. This study identified three key CPPs in a four step manufacturing process: (i) proper acidification during formation of the drug-lipid complexes (Step 1), (ii) liposome heat curing following liposomal particle sizing (Step 3), and (iii) flash-freezing at the initial stages of the lyophilization cycle (Step 4). Over-acidification led to rapid degradation of the drug, whereas under-acidification hampered full solubilization and formation of the soluble drug-lipid complexes. Extended heat treatment of the formed liposomes at 65 °C, just above the lipid phase transition temperature, brought dramatic changes in the aggregated state and/or packing of the drug in the liposomal bilayer, as followed by the complex changes in the UV/Vis spectra. Such thermal conditioning resulted in a five- to ten-fold reduction in the in-vitro toxicity of the drug product, bringing it close to the values for AmBisome used as control and measured by the RBC assay. Finally, flash-freezing conditions during lyophilization was critical to prevent aggregation and maintaining the 80-120 nm liposome size when reconstituted. Our research found that changes in the amphotericin's UV/Vis spectra were a sensitive CQA measure and provided a set of quantitative parameters for a facile non-destructive process monitoring in-situ, as well as for comparison of the quality of final formulations.

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Measurement of particle size and size distribution of complex drug products exhibiting complex rheological behaviors can be challenging as these properties may be beyond the theoretical assumptions of the measurement technique. Herein cyclosporine (CsA) ophthalmic emulsion was selected as a model complex system, and an in-depth assessment of particle size was performed using five fundamentally different particle sizing techniques, including dynamic light scattering (DLS), laser diffraction (LD), nanoparticle tracking analysis (NTA), cryogenic transmission electron microscopy (Cryo-TEM) and 2-dimensional diffusion ordered spectroscopy nuclear magnetic resonance (2D DOSY-NMR). The effect of various viscosity modifying and stabilizing excipients in the emulsions was assessed using four types of CsA formulations, i.e., 1) no viscosity modifying excipients, 2) carbomer copolymer type A (CCA), 3) Carbopol 1342, or 4) hydroxypropyl methyl cellulose (HMPC). In general, the variability of reported particle size increased, and is not as accurate, for emulsions dispersed in a non-Newtonian fluid and at higher emulsion concentrations. This effect was reduced in part by diluting the samples to lower volume fraction and a more Newtonian regime. To address the concern that sample dilution prior to measurement may induce physical instability in the emulsions, NTA was used to monitor average size at dilutions of up to 1:50,000. The size was found to remain constant and independent of the presence or type of stabilizer used. Cryo-TEM further confirmed that dilution did not alter particle size or morphology. Of the five evaluated techniques, Cryo-TEM and 2D DOSY NMR did not require dilution for measurement. The overestimate in DLS size measurements for certain CsA formulations was attributed to complex dispersant rheological behavior, particle-particle interactions, multiple light scattering events, and/or scattering interference from the polymers, which can be overcome by either testing under dilutions or by selecting one of the techniques less impacted by the interference of polymer.

The mechanism of drug release from complex dosage forms, such as multivesicular liposomes (MVLs), is complex and oftentimes sensitive to the release environment. This challenges the design and development of an appropriate in vitro release test (IVRT) method. In this study, a commercial bupivacaine MVL product was selected as a model product and an IVRT method was developed using a modified USP 2 apparatus in conjunction with reverse-dialysis membranes. This setup allowed the use of in situ UV-Vis probes to continuously monitor the drug concentration during release. In comparison to the traditional sample-and-separate methods, the new method allowed for better control of the release conditions allowing for study of the drug release mechanism. Bupivacaine (BPV) MVLs exhibited distinct tri-phasic release characteristics comprising of an initial burst release, lag phase and a secondary release. Temperature, pH, agitation speed and release media composition were observed to impact the mechanism and rate of BPV release from MVLs. The size and morphology of the MVLs as well as their inner vesicle compartments were analyzed using cryogenic-scanning electron microscopy (cryo-SEM), confocal laser scanning microscopy and laser diffraction, where the mean diameters of the MVLs and their inner "polyhedral" vesicles were found to be 23.6 ± 11.5 μm and 1.52 ± 0.44 μm, respectively. Cryo-SEM results further showed a decrease in particle size and loss of internal "polyhedral" structure of the MVLs over the duration of release, indicating erosion and rearrangement of the lipid layers. Based on these results a potential MVL drug release mechanism was proposed, which may assist with the future development of more biorelevant IVRT method for similar formulations.

Journal Article Distinguish Coexistence of Nanoemulsion and Liposome in Propofol by Cryogenic Transmission Electron Microscopy (cryo-TEM) Get access Yong Wu, Yong Wu Division of Biology, Chemistry and Materials Science, Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, Food and Drug Administration, Silver Spring, MD, United States Search for other works by this author on: Oxford Academic Google Scholar Peter Petrochenko, Peter Petrochenko Division of Biology, Chemistry and Materials Science, Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, Food and Drug Administration, Silver Spring, MD, United StatesDivision of Therapeutic Performance, Office of Research Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, United States Search for other works by this author on: Oxford Academic Google Scholar Ja Hye Myung, Ja Hye Myung Division of Biology, Chemistry and Materials Science, Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, Food and Drug Administration, Silver Spring, MD, United StatesDivision of Therapeutic Performance, Office of Research Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, United States Search for other works by this author on: Oxford Academic Google Scholar Soumyarwit Manna, Soumyarwit Manna Division of Biology, Chemistry and Materials Science, Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, Food and Drug Administration, Silver Spring, MD, United StatesDivision of Therapeutic Performance, Office of Research Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, United States Search for other works by this author on: Oxford Academic Google Scholar Bonhye Koo, Bonhye Koo Division of Biology, Chemistry and Materials Science, Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, Food and Drug Administration, Silver Spring, MD, United StatesDivision of Therapeutic Performance, Office of Research Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, United States Search for other works by this author on: Oxford Academic Google Scholar Stephanie Choi, Stephanie Choi Division of Therapeutic Performance, Office of Research Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, United States Search for other works by this author on: Oxford Academic Google Scholar Darby Kozak, Darby Kozak Division of Therapeutic Performance, Office of Research Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, United States Search for other works by this author on: Oxford Academic Google Scholar Jiwen Zheng Jiwen Zheng Division of Biology, Chemistry and Materials Science, Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, Food and Drug Administration, Silver Spring, MD, United States Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 23, Issue S1, 1 July 2017, Pages 852–853, https://doi.org/10.1017/S1431927617004925 Published: 04 August 2017

Porous PMMA is a versatile biomaterial with good biocompatibility but high susceptibility to bacterial colonization, which we mitigated by utilizing immobilized antimicrobial silver nanoparticles (AgNPs). A uniform porous thin film was deposited onto silicon wafers by simultaneously ablating PMMA and silver (Ag) using pulsed laser deposition (PLD) optimized for minimal human cell toxicity and antibacterial efficacy. PMMA without Ag became heavily colonized by E. coli in simulated dynamic conditions, while Ag-containing samples prevented all colonization. ICP-MS analysis demonstrated that the amount of leached Ag after 24h under simulated in vivo conditions (with serum media at 37°C and 5% CO2) increased in proportion to film thickness (and total silver content). 10,000, 14,000, and 20,000 laser pulse-deposited films released 0.76, 1.05, and 1.67μg/mL Ag, respectively, after 24h. Human bone marrow stromal cells (hBMSCs) grown directly on 10,000-pulse films (0.76μg/mL Ag released) for 24-h exhibited no cytotoxicity. Exposure to the remaining films produced cytotoxicity, necrosis, and apoptosis detected using flow cytometry. Examining both leachates and direct cell contact allowed us to develop an in vitro cytotoxicity test method and optimize a novel device material and coating to be nontoxic and bactericidal during both potential initial implantation and external use.

Journal Article Cryogenic Transmission Electron Microscopy (Cryo-TEM) Reveals Morphological Changes of Liposomal Doxorubicin during In Vitro Release Get access Yong Wu, Yong Wu Division of Biology, Chemistry and Materials Science, Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, Food and Drug Administration, Silver Spring, MD, United States Search for other works by this author on: Oxford Academic Google Scholar Peter Petrochenko, Peter Petrochenko Division of Biology, Chemistry and Materials Science, Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, Food and Drug Administration, Silver Spring, MD, United StatesOffice of Research Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, United States Search for other works by this author on: Oxford Academic Google Scholar Francis C Szoka, Francis C Szoka School of Pharmacy, University of California, San Francisco, CA Search for other works by this author on: Oxford Academic Google Scholar Soumyarwit Manna, Soumyarwit Manna Division of Biology, Chemistry and Materials Science, Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, Food and Drug Administration, Silver Spring, MD, United StatesOffice of Research Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, United States Search for other works by this author on: Oxford Academic Google Scholar Bonhye Koo, Bonhye Koo Division of Biology, Chemistry and Materials Science, Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, Food and Drug Administration, Silver Spring, MD, United StatesOffice of Research Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, United States Search for other works by this author on: Oxford Academic Google Scholar Nan Zheng, Nan Zheng Office of Research Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, United States Search for other works by this author on: Oxford Academic Google Scholar Wenlei Jiang, Wenlei Jiang Office of Research Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, United States Search for other works by this author on: Oxford Academic Google Scholar Jiwen Zheng Jiwen Zheng Division of Biology, Chemistry and Materials Science, Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, Food and Drug Administration, Silver Spring, MD, United States Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 23, Issue S1, 1 July 2017, Pages 1216–1217, https://doi.org/10.1017/S1431927617006742 Published: 04 August 2017

Studies M. S. Absar, S. Choi U.S. Food and Drug Administration Purpose Cyclosporine ophthalmic emulsion, 0.05%, is a topical immunomodulator which is indicated to increase tear production in patients whose tear production is presumed to be suppressed due to ocular infection associated with keratoconjunctivitis sicca. Currently, the FDA recommends two options to demonstrate bioequivalence (BE) for cyclosporine ophthalmic emulsion: (1) a clinical endpoint study or (2) in vitro studies with comparative physicochemical characterization. FDA posted a bioequivalence product-specific guidance on cyclosporine ophthalmic emulsion in June 2013, which was revised in February 2016 to include additional guidance on the conduct of the in vitro studies. A summary of scientific considerations when conducting studies to support the in vitro option will be presented. Methods For development of the in vitro option, a systematic approach was adopted to identify the physicochemical parameters that are critical for ocular bioavailability and BE of cyclosporine ophthalmic emulsion. In addition, the physicochemical properties of the reference product were determined by several different FDA laboratories to gain understanding of the drug product characteristics. Results Formulation excipients influence the critical physicochemical properties of ophthalmic emulsion products; hence, the test formulations should be qualitatively (Q1) and quantitatively (Q2) the same as the reference product. The globule size distribution (GSD) of cyclosporine emulsion can range from a few nanometers up to a micron depending on the instrument and sample preparation method used. Due to the inherent limitation of each GSD measurement technique, one single method may not be adequate to measure the entire size range. Therefore, complementary methods that are capable of measuring globules at the smaller and larger size ranges should be used. In addition, if the GSD is found to be multimodal, a high resolution analysis mode, i.e., narrow mode for laser diffraction (LD) and dynamic light scattering (DLS) methods should be used. GSD should be measured for undiluted samples and upon serial dilution, and the analysis mode, correlation curve (for DLS), dilution condition, dilution medium and histogram raw data should be reported. Since it is relatively difficult to control the extent of dilution for LD, an optimized %obscuration should be kept constant for all measurements to ensure similar concentrations of the test and reference products in the measurement beaker. For LD, the test and reference products can be statistically compared based on Dv,50 and SPAN. For DLS, intensity weighted size distribution, Di,50 and SPAN, along with harmonic intensity-weighted average particle diameter and polydispersity index can be reported to adequately represent the multimodal size distribution. D50 and SPAN should be evaluated using the population bioequivalence approach. In addition, equivalence between the test and RLD formulations in the shape of the GSD (such as the presence of multiple peaks) should be demonstrated by a suitable statistical method.

In an effort to develop new tools for diagnosing influenza in resource-limited settings, we fabricated a polycarbonate (PC)-polydimethylsiloxane (PDMS) hybrid microchip using a simple epoxy silica sol-gel coating/bonding method and employed it in sensitive detection of influenza virus with Europium nanoparticles (EuNPs). The incorporation of sol-gel material in device fabrication provided functionalized channel surfaces ready for covalent immobilization of primary antibodies and a strong bonding between PDMS substrates and PC supports without increasing background fluorescence. In microchip EuNP immunoassay (µENIA) of inactivated influenza viruses, replacing native PDMS microchips with hybrid microchips allowed the achievement of a 6-fold increase in signal-to-background ratio, a 12-fold and a 6-fold decreases in limit-of-detection (LOD) in influenza A and B tests respectively. Using influenza A samples with known titers, the LOD of influenza µENIA on hybrid microchips was determined to be ~10(4) TCID50 titer/mL and 10(3)-10(4) EID50 titer/mL. A comparison test indicated that the sensitivity of influenza µENIA enhanced using the hybrid microchips even surpassed that of a commercial laboratory influenza ELISA test. In addition to the sensitivity improvement, assay variation was clearly reduced when hybrid microchips instead of native PDMS microchips were used in the µENIA tests. Finally, infectious reference viruses and nasopharyngeal swab patient specimens were successfully tested using μENIA on hybrid microchip platforms, demonstrating the potential of this unique microchip nanoparticle assay in clinical diagnosis of influenza. Meanwhile, the tests showed the necessity of using nucleic acid confirmatory tests to clarify ambiguous test results obtained from prototype or developed point-of-care testing devices for influenza diagnosis.

1. Division of Biology, Chemistry and Materials Science, Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, Food and Drug Administration, Silver Spring, Maryland 20993, United States 2. Division of Therapeutic Performance, Office of Research Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993, United States

Understanding physicochemical properties of intravenous (IV) iron drug products is essential to ensure the manufacturing process is consistent and streamlined. The history of physicochemical characterization of IV iron complex formulations stretches over several decades, with disparities in iron core size and particle morphology as the major source of debate. One of the main reasons for this controversy is room temperature sample preparation artifacts, which affect accurate determination of size, shape and agglomeration/aggregation of nanoscale iron particles. The present study is first to report the ultra-fine iron core structures of four IV iron complex formulations, sodium ferric gluconate, iron sucrose, low molecular weight iron dextran and ferumoxytol, using a cryogenic transmission electron microscopy (cryo-TEM) preservation technique, as opposed to the conventional room temperature (RT-TEM) technique. Our results show that room temperature preparation causes nanoparticle aggregation and deformation, while cryo-TEM preserves IV iron colloidal suspension in their native frozen-hydrated and undiluted state. In contrast to the current consensus in literature, all four IV iron colloids exhibit a similar morphology of their iron oxide cores with a spherical shape, narrow size distribution and an average size of 2nm. Moreover, out of the four tested formulations, ferumoxytol exhibits a cluster-like community of several iron carbohydrate particles which likely accounts for its large hydrodynamic size of 25nm, measured with dynamic light scattering. Our findings outline a suitable method for identifying colloidal nanoparticle core size in the native state, which is increasingly important for manufacturing and design control of complex drug formulations, such as IV iron drug products.

The surface topographies of nanoporous anodic aluminum oxide (AAO) and titanium dioxide (TiO2) membranes have been shown to modulate cell response in orthopedic and skin wound repair applications. In this study, we: (1) demonstrate an improved atomic layer deposition (ALD) method for coating the porous structures of 20, 100, and 200 nm pore diameter AAO with nanometer-thick layers of TiO2 and (2) evaluate the effects of uncoated AAO and TiO2-coated AAO on cellular responses. The TiO2 coatings were deposited on the AAO membranes without compromising the openings of the nanoscale pores. The 20 nm TiO2-coated membranes showed the highest amount of initial protein adsorption via the micro bicinchoninic acid (micro-BCA) assay; all of the TiO2-coated membranes showed slightly higher protein adsorption than the uncoated control materials. Cell viability, proliferation, and inflammatory responses on the TiO2-coated AAO membranes showed no adverse outcomes. For all of the tested surfaces, normal increases in proliferation (DNA content) of L929 fibroblasts were observed over from 4 hours to 72 hours. No increases in TNF-alpha production were seen in RAW 264.7 macrophages grown on TiO2-coated AAO membranes compared to uncoated AAO membranes and tissue culture polystyrene (TCPS) surfaces. Both uncoated AAO membranes and TiO2-coated AAO membranes showed no significant effects on cell growth and inflammatory responses. The results suggest that TiO2-coated AAO may serve as a reasonable prototype material for the development of nanostructured wound repair devices and orthopedic implants.