Toxicity studies of highly bioavailable isoniazid loaded solid lipid nanoparticles as per Organisation for Economic Co-operation and Development (OECD) guidelines.

Solid lipid nanoparticles (SLNs) are presently being promoted to improve bioavailability of encapsulated drugs. These are well tolerated in living systems, as they are made from biocompatible material. Despite finding extensive applicability, these systems have not been sufficiently investigated for the toxicity so far. We have reported use of SLNs to improve plasma bioavailability of isoniazid (INH), a hepatotoxic, antitubercular drug. Presently we evaluate acute and repeated (28-day) oral dose toxicity, with satellite group, of developed INH loaded COMBI-SLN. In addition to high bioavailability, the COMBI-SLN exhibited 3 times higher LD50 (2000 mg/kg BW) versus 650 mg/kg BW for free INH. Results were complemented with histopathological evidence in brain, sciatic nerve and liver tissue all of which indicated enhanced safety of INH upon incorporation into SLNs. In the repeated dose study at doses selected as per Organisation for Economic Co-operation and Development (OECD) guidelines, a series of behavioural and haematological tests, clinical biochemistry (kidney and liver function, lipid profile) and histopathological studies were performed to evaluate the effect of low (250 mg/kg BW), medium (500 mg/kg BW) and high oral dose (1000 mg/kg BW). Absence of adverse effects like hepatotoxicity and peripheral neuropathy observed in rats at an oral intake level of 500 and 1000 mg/kg BW of COMBI-SLN, that is 20-40 folds above the anticipated human intake levels (after normalizing the surface area correction for rats), supports the conclusion that SLN are an intrinsically safe nanocarrier system that improves both the efficacy and the safety of INH.

Amphotericin B Loaded Nanostructured Lipid Carriers for Parenteral Delivery: Characterization, Antifungal and In vitro Toxicity Assessment.

BACKGROUND: Amphotericin B (AmB) is important for the treatment of systemic fungal infections. Nowadays, only intravenous administration (IV) of AmB has been available due to its low aqueous solubility. Two forms of AmB are available. The first is Fungizone®, a mixture of AmB and sodium deoxcycholate that produces severe nephrotoxicity. The second are lipid-based formulations that reduce nephrotoxicity, but they are costly and require higher dose than Fungizone®. Thus, a cheaper delivery system with reduced AmB toxicity is required. OBJECTIVE: To develop and characterize AmB loaded-nanostructured lipid carriers (AmB-loaded NLCs) for IV administration to reduce AmB toxicity. METHODS: AmB-loaded NLCs with different solid lipids were prepared by the high-pressure homogenization technique. Their physicochemical properties and the drug release profile were examined. The molecular structure of AmB, antifungal and hemolysis activities of developed AmB-loaded NLCs were also evaluated. RESULTS: AmB-loaded NLCs ~110 to ~140 nm in diameter were successfully produced with a zeta potential of ~-19 mV and entrapment efficiency of ~75%. In vitro release showed fast release characteristics. AmB-loaded NLCs could reduce the AmB molecular aggregation as evident from the absorbance ratio of the first to the fourth peak showing a partial aggregation of AmB. This result suggested that AmB-loaded NLCs could offer less nephrotoxicity compared to Fungizone®. In vitro antifungal activity of AmB-loaded NLCs showed a minimum inhibitory concentration of 0.25 µgmL-1. CONCLUSION: AmB-loaded NLCs present high potential carriers for effective IV treatment with prolonged circulation time and reduced toxicity.

A Comparative Assessment of the Aquatic Toxicity of Corexit 9500 to Marine Organisms.

The use of chemical dispersants during oil spill responses has long been controversial. During the Deepwater Horizon (DWH) oil spill, 1.8 million gallons of dispersant, mainly Corexit 9500, were applied in offshore waters to mitigate the human health and coastal environmental impact of surface oil contamination. To evaluate the potential impact of the dispersant on marine life, 18 species, representing important ecological and commercial taxa, were tested using low-energy, dispersant-only water accommodated fractions (WAFs) of Corexit 9500 and standard acute toxicity test methods. All prepared WAFs were analytically characterized. Analyses included the two dispersant markers found in the dispersant and evaluated in samples collected during the DWH Response, dioctylsulfosuccinate sodium salt, and dipropylene glycol n-butyl ether (DPnB). The median lethal and effective concentrations (LC/EC50s) were calculated using a nominal exposure concentration (mg/L, based on the experimental loading rate of 50 mg/L) and measured DPnB (µg/L). Results ranged from 5.50 to > 50 mg/L dispersant and 492 to > 304,000 µg/L DPnB. Species sensitivity distributions of the data demonstrated that taxa were evenly distributed; however, algae and oysters were among the more sensitive organisms. The calculated 5% hazard concentration (HC5) for DPnB (1172 µg/L) was slightly higher than the USEPA chronic criteria of 1000 µg/L and substantially higher than all measured concentrations of DPnB measured in the Gulf of Mexico during the DWH oil spill response.

Acute toxicity of Corexit EC9500A and assessment of dioctyl sulfosuccinate as an indicator for monitoring four oil dispersants applied to diluted bitumen.

The present study investigated oil dispersant toxicity to fish species typical of the cooler regions of Canada, together with less well-documented issues pertaining to oil dispersant monitoring. The oil dispersant toxicity of Corexit EC9500A was assessed for the freshwater fish species rainbow trout and the seawater species coho, chinook, and chum, with a final median lethal concentration (LC50) acute lethality range between 35.3 and 59.8 mg/L. The LC50 range was calculated using confirmed 0-h dispersant concentrations that were justified by fish mortality within the first 24 h of exposure and by variability of the dispersant indicator dioctyl sulfosuccinate (DOSS) used to monitor concentrations at later time points. To investigate DOSS as an oil dispersant indicator in the environment, microcosm systems were prepared containing Corexit EC9500A, Finasol OSR52, Slickgone NS, and Slickgone EW dispersants together with diluted bitumen. The DOSS indicator recovery was found to be stable for up to 13 d at 5 °C, 8 d at 10 °C, but significantly less than 8 d at ≥15 °C. After 3 d at temperatures ≥15 °C, the DOSS indicator recovery became less accurate and was dependent on multiple environmental factors including temperature, microbial activity, and aeration, with potential for loss of solvents and stabilizers. A final assessment determined DOSS to be a discrepant indicator for long-term monitoring of oil dispersant in seawater. Environ Toxicol Chem 2018;37:1309-1319. © 2018 SETAC.

Safety assessment of nanopesticides using the roundworm Caenorhabditis elegans.

The extensive use of pesticides is causing environmental pollution, affecting animal organisms in different habitats and also leading human health at risk. In this study, we present as an alternative the use of nanoparticles loaded with pesticides and report their toxicological assessment to a soil organism, Caenorhabditis elegans. Three nanoparticle formulations were analyzed: solid lipid nanoparticles loaded or not with atrazine and simazine, SLN; polymeric nanoparticles, NC_PCL loaded with atrazine; and chitosan/tripolyphosphate, CS/TPP, loaded or not with paraquat. All formulations, loaded or not with pesticides, increased lethality in a dose- dependent manner with similar LC50. Both loaded and unloaded NC_PCL were the most toxic formulations to developmental rate, significantly reducing worms length, even at low concentrations. In contrast, both CS/TPP nanoparticles were the least toxic, not affecting reproduction and body length at higher concentrations, probably due to the biocompatibility of chitosan. The physico-chemical characterization of nanoparticles after incubation in saline solution (used in exposure of organisms) has shown that these colloidal systems are stable and remain with the same initial characteristics, even in the presence of saline environment. Notably, our results indicate that the observed effects were caused by the nanoparticles per se. These results suggest that the development of nanoparticles aiming agriculture applications needs more studies in order to optimize the composition and then reduce their toxicity to non-target organisms.

Cytotoxicity assessment of lipid-based self-emulsifying drug delivery system with Caco-2 cell model: Cremophor EL as the surfactant.

PURPOSE: Caco-2 cells are used extensively for in vitro prediction of intestinal drug absorption. However, toxicity of excipients and formulations used can artificially increase drug permeation by damaging cell monolayers, thus providing misleading results. The present study aimed to investigate cytotoxicity of common lipid-based excipients and formulations on Caco-2 cells. METHODS: Medium-chain monoglycerides alone or in mixture with the surfactant Cremophor EL, with and without a medium-chain triglyceride, were prepared and incubated with Caco-2 cells from a series of culture stages with varying maturity. Cell viability was evaluated and cell membrane integrity assessed. RESULTS: Cytotoxicity of lipid-based formulations was influenced by the maturity of Caco-2 cells and formulation composition. One-day culture was most sensitive to lipids. When cultured for 5days, viability of Caco-2 cells was significantly improved. The 21-day Caco-2 monolayers maintained the highest survival rate. Microemulsion formulations exhibited significantly less cytotoxicity than neat lipids or surfactant at all stages of cell maturity, and microemulsions containing 1:1 mixtures of monoglyceride and triglyceride appeared to be best tolerated among all the formulations tested. Mechanistically, the observed cytotoxicity was partially due to lipid-induced rupture of cell membrane. CONCLUSIONS: Microemulsions of lipid-surfactant mixtures have less cytotoxicity than lipid alone. Maturity of Caco-2 cells renders significant resistance to cytotoxicity, and monolayers with 21-day maturity are more relevant to in vivo conditions and appear to be a more accurate in vitro model for cytotoxicity assessment.

The use of ephyrae of a scyphozoan jellyfish, Aurelia aurita, in the aquatic toxicological assessment of Macondo oils from the Deepwater Horizon incident.

Ephyrae of the scyphozoan jellyfish, Aurelia aurita, were evaluated in 96-hr acute toxicity tests for lethal response to Macondo crude oils from the Deepwater Horizon (DWH) incident in the Gulf of Mexico (GOM), Corexit 9500, and oil-dispersant mixtures. Water accommodated fractions (WAFs) of weathered and unweathered Macondo crude oils were not acutely toxic to ephyrae (LC50s > 100% WAF). The total PAHs (TPAHs), measured as the sum of 46 PAHs, averaged 21.1and 152 µg TPAH/L for WAFs of weathered and unweathered oil, respectively. Mortality was significantly (p = <0.0001) higher in the three highest exposure concentrations (184-736 µg TPAH/L) of chemically dispersed WAFs (CEWAF) compared to controls. Dispersant only tests resulted in a mean LC50 of 32.3 µL/L, which is in the range of previously published LC50s for marine zooplankton. Changes in appearance and muscle contractions were observed in organisms exposed to CEWAF dilutions of 12.5 and 25%, as early as 24 h post-exposure. Based on the results of these tests, crude oil alone did not cause significant acute toxicity; however, the presence of chemical dispersant resulted in substantial mortality and physical and behavioral abnormalities either due to an increase in hydrocarbons or droplet exposure.

Quantitative assessment of nanoparticle surface hydrophobicity and its influence on pulmonary biocompatibility.

To date, the role of nanoparticle surface hydrophobicity has not been investigated quantitatively in relation to pulmonary biocompatibility. A panel of nanoparticles spanning three different biomaterial types, pegylated lipid nanocapsules, polyvinyl acetate (PVAc) and polystyrene nanoparticles, were characterized for size, surface charge, and stability in biofluids. Surface hydrophobicity of five nanoparticles (50-150nm) was quantified using hydrophobic interaction chromatography (HIC) and classified using a purpose-developed hydrophobicity scale: the HIC index, range from 0.00 (hydrophilic) to 1.00 (hydrophobic). This enabled the relationship between the nanomaterial HIC index value and acute lung inflammation after pulmonary administration to mice to be investigated. The nanomaterials with low HIC index values (between 0.50 and 0.64) elicited little or no inflammation at low (22cm(2)) or high (220cm(2)) nanoparticle surface area doses per animal, whereas equivalent surface area doses of the two nanoparticles with high HIC index values (0.88-0.96) induced neutrophil infiltration, elevation of pro-inflammatory cytokines and adverse histopathology findings. In summary, a HIC index is reported that provides a versatile, discriminatory, and widely available measure of nanoparticle surface hydrophobicity. The avoidance of high (HIC index>~0.8) surface hydrophobicity appears to be important for the design of safe nanomedicines for inhalation therapy.

Gene expression and growth as indicators of effects of the BP Deepwater Horizon oil spill on spotted seatrout (Cynoscion nebulosus).

The BP Deepwater Horizon oil spill has great potential to negatively affect estuarine fish populations. In order to assess possible impacts of this event, a series of sublethal lab experiments were performed, using the economically and ecologically important species spotted seatrout (Cynoscion nebulosus). Larval and juvenile spotted seatrout were exposed to sublethal concentrations of high energy water accommodated fraction (HEWAF), chemically enhanced water accommodated fraction (CEWAF), or dispersant alone in an acute exposure. Response to exposure was evaluated with quantative polymerase chain reaction (qPCR) to examine expression of cytochrome P-4501A (CYP1A). Growth of larvae and juveniles over the duration of the experiment was measured as an index of physiological response. Our data showed that the different life stages respond differently to crude and dispersed oil, with larval spotted seatrout affected most by CEWAF, while juvenile spotted seatrout were affected to a greater extent by HEWAF. In both cases, the treatment with the highest CYP1A levels resulted in the greatest reductions in growth.

Lipid vesicles as membrane models for toxicological assessment of xenobiotics.

Traditionally animals and cell cultures have been used to assess the toxic potential of xenobiotics on cell membranes. In search for more reproducible, quantitative, cost- and time-effective assays, toxicologists have recently become interested in biomimetic lipid vesicle-based test systems. Lipid vesicles (liposomes) have long been appreciated as simple cell membrane models in biochemical and biophysical studies providing a good understanding of the physicochemical properties of liposome systems. More recently a number of reports have been published on the interactions of toxic substances with vesicles. Literature reports on liposome assays have appeared for widely different classes of xenobiotics, such as dental materials, antibiotics, detergents, and peptides. In this review we focus on those reports that contain a quantitative and significant correlation with more established toxicological tests like cell culture assays. We provide an introduction to the structure and main characteristics of vesicles and related lipid aggregates. The two main assays presented are leakage of fluorescence dyes and differential scanning calorimetry (DSC) measurements of the solid-ordered/liquid-disordered main phase transition temperature (Tm).