Action of the chemical agent fipronil on the reproductive process of semi-engorged females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae). Ultrastructural evaluation of ovary cells

The ovary of the tick Rhipicephalus sanguineus consists of a wall of epithelial cells and a large number of oocytes in five different developmental stages (I–V), which are attached to the wall by a pedicel. The present study provides ultrastructural information on the effects (dose–response) of the acaricide fipronil (Frontline^®) on ovaries of semi-engorged females of R. sanguineus, as well as it demonstrates some possible defense mechanisms used by oocytes to protect themselves against this chemical agent. Individuals were divided into four groups. Group I was used as control while groups II, III and IV were treated with fipronil at the concentrations of 1, 5 and 10 ppm, respectively. Fipronil at the concentration of 10 ppm had the strongest effect on the development of oocytes. At this concentration, even oocytes that reached the final developmental stage exhibited damaged cell structures. Moreover, the observation in fipronil-treated R. sanguineus ticks of damaged cellular components such as plasmic membrane, mitochondria and protein granules (due to alteration in the protein synthesis), and cellular defense mechanisms such as increase in the amount of cytoplasmic microtubules and large amounts of digestive vacuoles and myelin figures, were only possible by means of ultrastructure.     

Proteomic analysis of a model fish species exposed to individual pesticides and a binary mixture

Pesticides are nearly ubiquitous in surface waters of the United States, where they often are found as mixtures. The molecular mechanisms underlying the toxic effects of sub-lethal exposure to pesticides as both individual and mixtures are unclear. The current work aims to identify and compare differentially expressed proteins in brains of male fathead minnows (Pimephales promelas) exposed for 72 h to permethrin (7.5 μg/L), terbufos (57.5 μg/L) and a binary mixture of both. Twenty-four proteins were found to be differentially expressed among all three treatments relative to the control using an ANOVA followed by a Dunnett's post hoc test (p ≤ 0.05). One protein was found to be differentially expressed among all treatment groups and one protein was in common between the terbufos and the mixture group. Fifteen spots were successfully sequenced using LC-MS/MS sequencing. Proteins associated with the ubiquitin-proteasome system, glycolysis, the cytoskeleton and hypoxia were enriched. As a second objective, we attempted to establish protein expression signatures (PES) for individual permethrin and terbufos exposures. We were unable to generate a useable PES for terbufos; however, the permethrin PES was able to distinguish between control and permethrin-exposed individuals in an independent experiment with an accuracy of 87.5%. This PES also accurately classified permethrin exposed individuals when the exposure occurred as part of a mixture. The identification of proteins differentially expressed as a result of pesticide exposure represent a step forward in the understanding of mechanisms of toxicity of permethrin and terbufos. They also allow a comparison of molecular responses of the binary mixture to single exposures. The permethrin PES is the first step in establishing a method to determine exposures in real-world scenarios.     

Protective effects of a polysaccharide from Hizikia fusiformis against ethanol toxicity in rats

Hizikia fusiformis is an edible brown alga that is widely consumed in Korea, Japan, and China and possesses a number of potentially beneficial compounds, including antioxidants and anticoagulants. No reports have investigated potential H. fusiformis protectants against ethanol-induced peptic injury. We extracted a polysaccharide from H. fusiformis (Hf–PS-1) that exhibited protective effects against ethanol-induced peptic injury and related mechanisms in rats. Experimental animals were divided into three groups: control, ethanol-only, and ethanol + Hf–PS-1. The ethanol-only group exhibited decreased levels of total glutathione (GSH) and increased levels of jun N-terminal kinase (JNK) phosphorylation relative to the control group, whereas levels were significantly increased and decreased, respectively, in the ethanol + Hf–PS-1 group. The ethanol-only group also exhibited increased levels of extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation relative to the control group; these levels were not significantly different in the ethanol + Hf–PS-1 group. Hf–PS-1 appeared to reduce ethanol-induced gastric injury. Therefore, we suggest that Hf–PS-1 could protect against ethanol-induced peptic ulcers primarily through a mechanism associated with the inhibition of JNK activation.     

Changes in correlation coefficients of exposure markers as a function of intensity of occupational exposure to toluene

This study was initiated to identify a marker of choice to monitor occupational exposure to toluene through quantitative evaluation of changes in correlation coefficients (CCs), taking advantage of a large database. Six known or proposed exposure markers in end-of-shift blood (B) and urine (U) were studied, i.e., toluene in blood (Tol-B) and benzyl alcohol, benzylmercapturic acid, o-cresol, hippuric acid and toluene in urine (BeOH-U, BMA-U, o-CR-U, HA-U, and Tol-U, respectively). To construct a database, data on 8-h time-weighted average intensity of occupational exposure to toluene and resulting levels of the six exposure markers in blood or urine were cited for 901 cases from previous four publications of this study group and combined with 146 new cases. In practice, 874 cases (all men) were available when extremely dilute or dense urine samples were excluded. The 874 cases were classified taking the upper limit (from 120 ppm to 1 ppm) of the toluene exposure concentration, and the CCs for the six markers with TWA toluene exposure intensity were calculated. For further evaluation, the 874 cases were divided into 10%-tiles in terms of TWA toluene exposures, and several 10%-tiles were combined so that sufficient numbers of cases were available for calculation of the CCs at various levels of toluene exposure. Perusal was made to know whether or not and which one of the six makers gave significant CC even at low level of toluene exposure. The CCs for BMA-U, o-CR-U and HA-U with TWA toluene exposure were well >0.7 when toluene exposure was intense (e.g., up to 60–100 ppm as the upper limit of the exposure range), but were reduced when the upper limit of toluene exposure was less than 50 ppm, and the CCs were as small as ≦0.2 when the upper limit was about 10 ppm or less. In contrast, Tol-U and Tol-B were correlated with exposure to toluene down to the ≦3 ppm range. The CC for BeOH-U was <0.1 almost throughout the exposure ranges. Further analyses showed that the CCs for all markers (except the CC for BeOH-U) were >0.4 when the cases in the 60th–100th%-tiles were examined. The CCs for Tol-U and Tol-B were >0.3 also for cases in the 0th–60th or 30th–70th%-tiles, whereas the CCs for other four markers were <0.3. In over-all evaluation, it was concluded that HA and o-CR are among the markers of choice to monitor occupational toluene exposure at high levels, and that only un-metabolized toluene in urine or in blood is recommended when toluene exposure level is low (e.g., 10 ppm or less). Toluene in urine may be preferred rather than that in blood due to practical reasons, such as non-invasiveness.     

Safety evaluation of a high-lipid algal biomass from Chlorellaprotothecoides

Chlorella are traditionally freshwater green algae that have been evaluated for dietary purposes because of their nutritional value. This study investigates the safety of Chlorella protothecoides in a 28-day study. Sprague–Dawley rats were administered 0 (control), 2.5, 5.0, or 10% of their diet for 28 days using an FDA Redbook protocol. The average daily dietary intake of algal biomass was determined to be 0, 1794, 3667, and 7557 mg/kg body weight for males and 0, 1867, 3918, and 8068 mg/kg body weight for females. Hematological and biochemical analyses were conducted, and upon completion, gross and microscopic evaluations were performed. No signs of toxicity were observed. Although statistically significant alterations were noted in several parameters among males and females, these changes were deemed to be of no toxicological significance due to the lack of dose–response relationships, the fact that they occurred in only one sex, and the lack of any supporting gross or microscopic alterations. The no-observed-adverse-effect level for the algal biomass under the conditions of this study was considered to be 10% in the diet, the highest dose tested.     

The antioxidative effect of Iranian Mentha pulegium extracts and essential oil in sunflower oil

The aim of the present study was to evaluate antioxidative activities of the essential oil, methanol and water extracts of Iranian pennyroyal in vegetable oil during storage. Different concentrations (0, 200, 400, 600, 800 and 1000 ppm) of essential oil, water and methanol extracts and beta-hydroxy toluene (BHT; 200 ppm) were added to sunflower oil emulsion in the presence of cupric ions and incubated for 7 days at 60 °C. Peroxide values (PVs) and thiobarbituric acid reacting substances (TBARS) levels were measured in each day up to day of seven. Furthermore, antioxidant capacity of the essential oil and extracts were determined using DPPH and β-carotene–linoleic acid methods. Values were compared among groups in each incubation time points using ANOVA. Results showed that DPPH and β-carotene–linoleic acid assay findings on the Mentha pulegium extracts were comparable to those found on BHT. Furthermore, in all incubation time points, M. pulegium extracts lowered PVs and TBARS levels when compared to the control (p < 0.001). In this respect, water extract was more potent than the methanol extract. Essential oil did not show considerable antioxidative effect. It seems that water extract of M. pulegium is a potent antioxidant which makes it as a potential antioxidant for oil and oily products during storage.     

Chemical profile,antifungal, antiaflatoxigenic and antioxidant activity of Citrus maxima Burm. and Citrus sinensis (L.) Osbeck essential oils and their cyclic monoterpene, dl-limonene

The study deals with antifungal, antiaflatoxigenic and antioxidant activity of Citrus maxima and Citrus sinensis essential oils (EOs) and their phytochemical composition. The EOs were obtained by hydrodistillation and their chemical profile was determined through GC and GC–MS analysis. Both the EOs and their 1:1 combination showed broad fungitoxic spectrum against different food contaminating moulds. The EOs and their combination completely inhibited aflatoxin B₁ (AFB₁) production at 500 ppm, whereas, dl-limonene, the major component of EOs showed better antiaflatoxigenic efficacy even at 250 ppm. Both the oils exhibited antioxidant activity as DPPH free radical scavenger in dose dependent manner. The IC₅₀ for radical scavenging efficacy of C. maxima and C. sinensis oils were to be 8.84 and 9.45 μl ml⁻¹, respectively. The EOs were found non-mammalian toxic showing high LD₅₀ for mice (oral, acute). The oils may be recommended as safe plant based antimicrobials as well as antioxidants for enhancement of shelf life of food commodities by checking their fungal infestation, aflatoxin production as well as lipid peroxidation.     

Dietary administration of paraquat for 13 weeks does not result in a loss of dopaminergic neurons in the substantia nigra of C57BL/6J mice

Several investigations have reported that mice administered paraquat dichloride (PQ·Cl₂) by intraperitoneal injection exhibit a loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). In this study, male and female C57BL/6J mice were administered PQ·Cl₂ in the diet at concentrations of 0 (control), 10, and 50 ppm for a duration of 13 weeks. A separate group of mice were administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) during week 12 as positive controls to produce a loss of dopaminergic neurons in the SNpc. The comparative effects of PQ and MPTP on the SNpc and/or striatum were assessed using neurochemical, neuropathological, and stereological endpoints. Morphological and stereological assessments were performed by investigators ‘blinded’ to the origin of the tissue. Neither dose of PQ·Cl₂ (10 or 50 ppm in the diet) caused a loss of striatal dopamine or dopamine metabolite concentrations in the brains of mice. Pathological assessments of the SNpc and striatum showed no evidence of neuronal degeneration or astrocytic/microglial activation. Furthermore, the number of tyrosine hydroxylase-positive (TH⁺) neurons in the SNpc was not reduced in PQ-treated mice. In contrast, MPTP caused a decrease in striatal dopamine concentration, a reduction in TH⁺ neurons in the SNpc, and significant pathological changes including astrocytic and microglial activation in the striatum and SNpc. The MPTP-induced effects were greater in males than in females. It is concluded that 13 weeks of continuous dietary exposure of C57BL/6J mice to 50 ppm PQ·Cl₂ (equivalent to 10.2 and 15.6 mg PQ ion/kg body weight/day for males and females, respectively) does not result in the loss of, or damage to, dopaminergic neurons in the SNpc.     

Injury effects of ginkgolide B on maturation of mouse oocytes,fertilization, and fetal development in vitro and in vivo

Ginkgolide B (GKB), the major active component of Ginkgo biloba extracts, exerts both stimulatory and inhibitory effects on apoptotic signaling. Previous studies by our group demonstrated that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell number, hinders early postimplantation blastocyst development, and increases early-stage blastocyst death. Here, we further investigate the effects of GKB on oocyte maturation, and subsequent pre- and postimplantation development in vitro and in vivo. In our experiments, GKB induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with 1–6 μM GKB during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased placental and fetal weights. Data obtained using an in vivo mouse model further disclosed that consumption of drinking water containing 3–6 μM GKB led to decreased oocyte maturation and in vitro fertilization, as well as early embryo developmental injury, specifically, inhibition of development to the blastocyst stage in vivo. To our knowledge, this is the first study to investigate the impact of GKB on maturation of mouse oocytes, fertilization, and sequential embryonic development.     

Safety evaluation of fibermalt

Fibermalt is a new soluble fiber food ingredient produced with the use of an alternansucrase enzyme from Leuconostoc mesenteroides expressed in a non-pathogenic strain of Escherichia coli. Fibermalt is predominantly composed of indigestible maltose alternan oligosaccharides (?80%). Fibermalt was non-mutagenic in a bacterial reverse mutation test. In a 13-week dietary rat study, fibermalt was administered at 0 (control), 50,000, 100,000 or 150,000 ppm. Statistically significant increases in food consumption were generally observed throughout the study in males receiving 100,000 or 150,000 ppm and in females receiving 100,000 ppm. However, there was no effect of fibermalt on mean body weight, body weight gain or food efficiency. All animals survived to scheduled termination and no adverse clinical signs were attributed to administration of fibermalt. There were no toxicologically relevant changes in hematology, clinical chemistry or urinalysis parameters or organ weights in males or females ingesting any concentration of fibermalt. Any macroscopic or microscopic findings were considered incidental, of normal variation and/or of minimal magnitude for test substance association. Based on these results, fibermalt is not mutagenic as evaluated in a bacterial reverse mutation test and has an oral subchronic (13-week) no observable adverse effect level (NOAEL) of 150,000 ppm in rats.     

Acute and subacute toxicity of yeast hydrolysate from Saccharomyces cerevisiae

The objective of this study was to obtain data on the safety-in-use of yeast hydrolysate in 10–30 kDa molecular weight as a dietary supplement by assessing its acute and subacute oral toxicity in female and male Sprague–Dawley (SD) rats. The single oral dose of the hydrolysate at 5000 mg/kg did not produce mortality or significant changes in the general behavior and gross appearance of the internal organs of rats. In subacute toxicity study, the hydrolysate was administered orally at a dose of 1000 mg/kg/day for a period of 14 days. The satellite group was treated with the hydrolysate at the same dose and the same period and kept for another 14 days after treatment. There were no significant differences in organ weights between control and treated group of both sexes. Hematological analysis and blood chemistry revealed no toxicity effects of Saccharomyces cerevisiae hydrolysate. Pathologically, neither gross abnormalities nor histopathological changes were observed. These results show that the hydrolysate possesses very low toxicity as indicated in SD rat model.     

Taraxerone enhances alcohol oxidation via increases of alcohol dehyderogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities and gene expressions

The present study, taraxerone (d-friedoolean-14-en-3-one) was isolated from Sedum sarmentosum with purity 96.383%, and its enhancing effects on alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities were determined: EC₅₀ values were 512.42 ± 3.12 and 500.16 ± 3.23 μM for ADH and ALDH, respectively. In order to obtain more information on taraxerone related with the alcohol metabolism, 40% ethanol (5 mL/kg body weight) with 0.5–1 mM of taraxerone were administered to mice. The plasma alcohol and acetaldehyde concentrations of taraxerone-treated groups were significantly lowered than those of the control group (p < 0.01): approximately 20–67% and 7–57% lowered for plasma alcohol and acetaldehyde, respectively. Compare to the control group, the ADH and ALDH expressions in the liver tissues were abruptly increased in the taraxerone-treated groups after ethanol exposure. In addition, taraxerone prevented catalase, superoxide dismutase, and reduced glutathione concentrations from the decrease induced by ethanol administration with the concentration dependent manner.     

Role of Panax ginseng as an antioxidant after cadmium-induced hepatic injuries

Liver, being primary site for biotransformation of foreign compounds is vulnerable to various chemical assaults. Ginseng has a wide range of pharmacological and therapeutical action. In the present study an attempt has been made to study the cadmium chloride (CdCl₂) induced toxicity in liver and its possible protection by Panax ginseng. Swiss albino mice were divided into four groups: (i) Control group – only vehicle (double distilled water) (ii) Ginseng treated group – 10 mg/kg b.wt. orally (iii) CdCl₂ treated group – 1.0 mg/kg b.wt. CdCl₂ i.p. (iv) Combination group – Ginseng root extract (10 mg/kg b.wt.) and CdCl₂ (1.0 mg/kg b.wt.). Activities of alkaline phosphatase, GOT, GPT were measured in serum and lipid peroxidation (LPO) and GSH content were measured in liver. The results indicated a significant increase in LPO, GOT, GPT activities and decrease in GSH and serum alkaline phosphatase activities after CdCl₂ treatment. Ginseng alone did not show any significant alterations except a significant decrease in LPO level. Combined treatment of Ginseng and CdCl₂ showed significant decrease in LPO, GOT, GPT and elevation in GSH and serum alkaline phosphatase as compared to CdCl₂ treated group. Thus, Ginseng is found to be protective against cadmium-induced hepatic injuries.     

Safety assessment of heat-sterilized green tea catechin preparation: A 6-month repeat-dose study in rats

Evidence suggests that the purported health benefits associated with green tea consumption are related to tea catechins. In the present study, potential adverse effects of a standardized heat-sterilized green tea catechin (GTC-H) preparation was investigated following gavage administration to rats at doses of 0, 120, 400, 1200 mg/kg/day for 6 months. A decaffeinated high-dose group (1200 mg/kg/day) (GTC–HDC), was included for comparison. A possibly test article-related clinical finding of intermittent increased activity was noted in the 400 and 1200 mg/kg/day GTC-H groups, but was not considered to be adverse. Lower body weight gains without any decrease in food consumption were noted in the high-dose (1200 mg/kg/day)-treated GTC-H and GTC–HDC females. In the high-dose male GTC-H group, a lower total motor activity count for the 60-min session was noted prior to dosing at the study week 25 evaluations compared to the control group. Similar changes were not observed in the GTC–HDC group. Based on the results of this study, the no-observed-adverse-effect level (NOAEL) for GTC-H was 1200 mg/kg/day for males, the highest dose tested, and 400 mg/kg/day for females based on reduced body weight gains. The NOAEL for GTC–HDC was 1200 mg/kg/day for males and could not be determined in females.     

Effects of acute in vitro exposure of murine precision-cut lung slices to gaseous nitrogen dioxide and ozone in an air–liquid interface (ALI) culture

The aim of this study was to establish an air–liquid interface (ALI) culture of precision-cut lung slices (PCLS) for direct exposure of lung cells to gaseous contaminants. Nitrogen dioxide (NO₂) and ozone (O₃) were selected as model gas compounds. Acute pro-inflammatory and toxic effects of NO₂ and O₃ on live lung tissue were investigated. Murine PCLS were exposed to different flow rates (3–30 mL/min) of synthetic air, O₃ (3.5–8.5 ppm), or NO₂ (1–80 ppm). Tissue survived ex vivo in ALI culture and resisted exposure to NO₂ (1–10 ppm) and O₃ (3.5–8.5 ppm) for 1 h. Longer exposure to NO₂ resulted in a clear loss of viability, whereas exposure to O₃ was less effective. Exposure to NO₂ dose-dependently induced release of the pro-inflammatory IL-1α (40%), whereas RANTES, IL-12, and eotaxin remained unchanged. Early secretion of IL-1α (80%), RANTES (>800%), MIP-1β (44%), and MCP-1 (60%) was already detected after 1 h of exposure to O₃. The obtained data showed that direct exposure to O₃ and NO₂ induced cytotoxicity and pro-inflammatory responses in PCLS with ALI culture. This provides a model that more closely resembles in vivo exposure of airborne contaminants, and thus should be appropriate for toxicity testing.     

Essential oil composition and antioxidant activities of Curcuma aromatica Salisb.

The chemical composition of the hydro-distilled essential oil from leaves of Curcuma aromatica Salisb. was analysed by GC–MS. Twenty-three compounds representing 94.29% of the total oil were identified. The antioxidant activities of the oil and various extracts of C. aromatica were evaluated by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radical-scavenging assays. The oil and methanol extract showed potent DPPH radical-scavenging activities (IC₅₀ = 14.45 and 16.58 μg/ml, respectively), which were higher than butylated hydroxyanisole (IC₅₀ = 18.27 μg/ml). The extracts also exhibited remarkable superoxide radical-scavenging activities (IC₅₀ = 22.6–45.27 μg/ml) and the activity in the methanol extract was superior to all other extracts (IC₅₀ = 22.6 μg/ml). Furthermore, the amount of total phenolic compounds was determined and its content in ethyl acetate extract was the highest as compared to other extracts. The results indicate that the oil and extracts of C. aromatica could serve as an important bio-resource of antioxidants for using in the food industries.     

Chemical composition,in vitro anti-microbial,antifungal and antioxidant activities of the essential oil and methanolic extract of Hymenocrater longiflorus Benth., of Iran

In this study we identified the chemical composition, anti-microbial and antioxidant effects of essential oil and methanolic extract of Hymenocrater longiflorus Benth. Totally 87 volatile compounds from the essential oil in H. longiflorus, were identified by gas chromatography–mass spectrometry (GC–MS). These compounds are mainly monoterpene hydrocarbons, sesquiterpene hydrocarbons, oxygenated monoterpenes and oxygenated sesquiterpenoids compounds. The anti-microbial and antifungal activity of plants extracts against several pathogenic microorganisms was studied by disc diffusion and minimum inhibitory concentration procedures. The results revealed that the essential oil and polar sub-fraction are effective mostly against Staphylococcus aureus, Aspergillus niger and Candida albicans. The antioxidant activity was also determined by 1,1′-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging, β-carotene linoleic acid assay and reducing power. In addition the total phenol of essential oil (54.6 ± 1.2), polar sub-fraction (50.0 ± 1.4) and non-polar sub-fraction (64.7 ± 2.0) were determined.     

Protective effects of Etlingera elatior extract on lead acetate-induced changes in oxidative biomarkers in bone marrow of rats

Several environmental toxins with toxic effects to the bone marrow have been identified. Pathology associated with lead intoxication is due to the cellular damage mediated by free radicals. In the current study, we examined the effect of Etlingera elatior extract on lead-induced changes in the oxidative biomarkers and histology of bone marrow of rats. Sprague–Dawley rats were exposed to 500 ppm lead acetate in their drinking water for 14 days. E. elatior extract was treated orally (100 mg/kg body weight) in combination with, or after lead acetate treatment. The results showed that there was a significant increase in lipid hydroperoxide, protein carbonyl content and a significant decrease in total antioxidants, super oxide dismutase, glutathione peroxidase and glutathione – S-transferase in bone marrow after lead acetate exposure. Treatment with E. elatior decreased lipid hydroperoxides and protein carbonyl contents and significantly increased total antioxidants and antioxidant enzymes. Treatments with E. elatior extract also reduced, lead-induced histopathological damage in bone marrow. In conclusion, these data suggest that E. elatior has a powerful antioxidant effect, and it protects the lead acetate-induced bone marrow oxidative damage in rats.     

Development and validation of a rapid reversed-phase HPLC method for the determination of the non-nucleoside reverse transcriptase inhibitor dapivirine from polymeric nanoparticles

The objective of this work was to develop and validate a rapid reversed-phase (RP) high-performance liquid chromatography (HPLC) method for the in vitro pharmaceutical characterization of dapivirine-loaded polymeric nanoparticles. Chromatographic runs were performed on a RP C18 column with a mobile phase comprising acetonitrile–0.5% (w/v) triethanolamine solution in isocratic mode (80:20, v/v) at a flow rate of 1 ml/min. Dapivirine was detected at a wavelength of 290 nm. The method was shown to be specific, linear in the range of 1–50 μg/ml (R² = 0.9998), precise at the intra-day and inter-day levels as reflected by the relative standard deviation values (less than 0.85%), accurate (recovery rate of 100.17 ± 0.35%), and robust to changes in the mobile phase and column brand. The detection and quantitation limits were 0.08 and 0.24 μg/ml, respectively. The method was successfully used to determine the loading capacity and association efficiency of dapivirine in poly(lactic-co-glycolic acid)-based nanoparticles and its in vitro release.