Mitigating amphotericin B cytotoxicity through gliadin-casein nanoparticles: Insights into synthesis, optimization, characterization, in vitro release and cytotoxicity evaluation.

Amphotericin B (AmB) is a widely used antifungal agent; however, its clinical application is limited due to severe side effects and nephrotoxicity associated with parenteral administration. In recent years, there has been growing interest in the utilization of food-grade materials as innovative components for nanotechnology-based drug delivery systems. This study introduces gliadin/casein nanoparticles encapsulating AmB (AmB_GliCas NPs), synthesized via antisolvent precipitation. Formulation was refined using a 24 factorial design, assessing the influence of gliadin and casein concentrations, as well as organic and aqueous phase volumes, on particle size, polydispersity index (PDI), and zeta potential. The optimal composition with 2 % gliadin, 0.5 % casein, and a 1:5 organic-to-aqueous phase ratio, yielded nanoparticles with a 442 nm size, a 0.307 PDI, a -20 mV zeta potential, and 82 % entrapment efficiency. AmB was confirmed to be amorphous within the nanoparticles by X-ray diffraction. These NPs released AmB sustainably over 96 h, primarily in its monomeric form. Moreover, NPs maintained stability in simulated gastrointestinal fluids with minimal drug release and showed significantly lower hemolytic activity and cytotoxicity on Vero cells than free AmB, suggesting their promise for oral AmB delivery.

Nanotechnology-Based Approaches for Voriconazole Delivery Applied to Invasive Fungal Infections.

Invasive fungal infections increase mortality and morbidity rates worldwide. The treatment of these infections is still limited due to the low bioavailability and toxicity, requiring therapeutic monitoring, especially in the most severe cases. Voriconazole is an azole widely used to treat invasive aspergillosis, other hyaline molds, many dematiaceous molds, Candida spp., including those resistant to fluconazole, and for infections caused by endemic mycoses, in addition to those that occur in the central nervous system. However, despite its broad activity, using voriconazole has limitations related to its non-linear pharmacokinetics, leading to supratherapeutic doses and increased toxicity according to individual polymorphisms during its metabolism. In this sense, nanotechnology-based drug delivery systems have successfully improved the physicochemical and biological aspects of different classes of drugs, including antifungals. In this review, we highlighted recent work that has applied nanotechnology to deliver voriconazole. These systems allowed increased permeation and deposition of voriconazole in target tissues from a controlled and sustained release in different routes of administration such as ocular, pulmonary, oral, topical, and parenteral. Thus, nanotechnology application aiming to delivery voriconazole becomes a more effective and safer therapeutic alternative in the treatment of fungal infections.

Polyethylene Glycol-Stabilized Zein Nanoparticles Containing Gallic Acid.

Research background: Gallic acid is a polyphenol with antioxidant and antitumor activities; however, its use as a nutraceutical or drug is hindered by its low bioavailability. Zein is a natural protein found in corn and has been applied as nanoparticle drug carrier. In this study, zein nanoparticles were obtained and stabilized with polyethylene glycol (PEG) as gallic acid carriers. Experimental approach: Nanoparticles were obtained by the liquid-liquid method and characterized in terms of mean size, polydispersity index, zeta potential, morphology, solid-state interactions and encapsulation efficiency/drug loading. The stability of nanoparticles was evaluated in simulated gastrointestinal fluids and food simulants, and the antioxidant activity was determined by the scavenging of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. Results and conclusions: Zein nanoparticles containing gallic acid were obtained and stabilized only in the presence of PEG. Under optimal conditions, nanoparticles with mean size <200 nm, low polydispersity index (<0.25) and negative zeta potential (-20 mV) were obtained. The gallic acid encapsulation efficiency was about 40%, loading about 5%, and it was encapsulated in an amorphous state. Fourier transform infrared spectroscopy (FTIR) did not identify chemical interactions after gallic acid nanoencapsulation. Zein nanoparticles were more prone to release the gallic acid in gastric than intestinal simulated medium; however, more than 50% of drug content was protected from premature release. In food simulants, the gallic acid release from nanoparticles was prolonged and sustained. Moreover, the nanoencapsulation did not reduce the antioxidant activity of gallic acid. Novelty and scientific contribution: The results show the importance of PEG in the formation and its effect on the properties of zein nanoparticles obtained by the liquid-liquid dispersion method. This study indicates that PEG-stabilized zein nanoparticles are potential carriers for oral intake of gallic acid, preserving its antioxidant properties and enabling its use in the pharmaceutical and food industries.

Chitosan modified poly (lactic acid) nanoparticles increased the ursolic acid oral bioavailability.

Ursolic acid (UA) is a naturally occurring triterpene that has been investigated for its antitumor activity. However, its lipophilic character hinders its oral bioavailability, and therapeutic application. To overcome these limitations, chitosan (CS) modified poly (lactic acid) (PLA) nanoparticles containing UA were developed, characterized, and had their oral bioavailability assessed. The nanoparticles were prepared by emulsion-solvent evaporation technique and presented a mean diameter of 330 nm, zeta potential of +28 mV, spherical shape and 90% encapsulation efficiency. The analysis of XRD and DSC demonstrated that the nanoencapsulation process induced to UA amorphization. The in vitro release assay demonstrated that 53% of UA was released by diffusion after 144 h, following a second-order release kinetics. In simulated gastrointestinal fluids and mucin interaction tests, CS played an important role in stability and mucoadhesiveness improvement of PLA nanoparticles, respectively. In the presence of erythrocytes, nanoparticles proved their hemocompatibility. In tumor cells, nanoparticles presented lower cytotoxicity than free UA, due to slow UA release. After a single oral dose in rats, CS modified PLA nanoparticles increased the UA absorption, reduced its clearance and elimination, resulting in increased bioavailability. The results show the potential application of these nanoparticles for UA oral delivery for cancer therapy.

Chitosan Nanoparticles Potentiate the in vitro and in vivo Effects of Curcumin and other Natural Compounds.

The development of biodegradable nanoparticles is an important tool for the biological transport of chemical compounds. The nanoencapsulation reduces the biopharmaceutical and pharmacokinetic drawbacks of compounds and enhances their biological properties. Naturally occurring polymers such as proteins and polysaccharides have been widely applied in the development of nanostructured systems of several therapeutic agents. Among them is chitosan, a crustacean-carapace-chitin derived biopolymer. In addition to its biocompatibility and biodegradability, chitosan is known for its mucoadhesion properties. Chitosan-based nanostructured systems potentiate most of the aspects of the loaded drugs, including cellular transport and other biological effects. The use of chitosan nanoparticles enhances permeation, stability, and bioactivity of natural compounds. In this review, an overview of the main features of chitosan nanoparticles that improved in vitro and in vivo effects of bioactive natural molecules is given, emphasizing the results obtained with curcumin.

Nanoparticles as a Tool for Broadening Antifungal Activities.

Fungal infections are diseases that are considered neglected although their infection rates have increased worldwide in the last decades. Thus, since the antifungal arsenal is restricted and many strains have shown resistance, new therapeutic alternatives are necessary. Nanoparticles are considered important alternatives to promote drug delivery. In this sense, the objective of the present study was to evaluate the contributions of newly developed nanoparticles to the treatment of fungal infections. Studies have shown that nanoparticles generally improve the biopharmaceutical and pharmacokinetic characteristics of antifungals, which is reflected in a greater pharmacodynamic potential and lower toxicity, as well as the possibility of prolonged action. It also offers the proposition of new routes of administration. Nanotechnology is known to contribute to a new drug delivery system, not only for the control of infectious diseases but for various other diseases as well. In recent years, several studies have emphasized its application in infectious diseases, presenting better alternatives for the treatment of fungal infections.

COVID-19: The question of genetic diversity and therapeutic intervention approaches.

Coronavirus disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2), is the largest pandemic in modern history with very high infection rates and considerable mortality. The disease, which emerged in China's Wuhan province, had its first reported case on December 29, 2019, and spread rapidly worldwide. On March 11, 2020, the World Health Organization (WHO) declared the COVID-19 outbreak a pandemic and global health emergency. Since the outbreak, efforts to develop COVID-19 vaccines, engineer new drugs, and evaluate existing ones for drug repurposing have been intensively undertaken to find ways to control this pandemic. COVID-19 therapeutic strategies aim to impair molecular pathways involved in the virus entrance and replication or interfere in the patients' overreaction and immunopathology. Moreover, nanotechnology could be an approach to boost the activity of new drugs. Several COVID-19 vaccine candidates have received emergency-use or full authorization in one or more countries, and others are being developed and tested. This review assesses the different strategies currently proposed to control COVID-19 and the issues or limitations imposed on some approaches by the human and viral genetic variability.

Intranasal administration of perillyl alcohol-loaded nanoemulsion and pharmacokinetic study of its metabolite perillic acid in plasma and brain of rats using ultra-performance liquid chromatography/tandem mass spectrometry.

Perillyl alcohol (POH) is a monocyclic terpene that has strong antitumor activity. Brain tumors are particularly difficult to treat with therapeutic agents, and clinical trials have shown their low tolerance through oral administration. We proposed the entrapment of POH into an oil-in-water chitosan nanoemulsion aiming its intranasal administration for brain targeting. An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantitation of total metabolite perillic acid (PA) in plasma and brain of rats. The rat samples containing the metabolite were treated by liquid-liquid extraction with acetonitrile. The mobile phase was 0.1% formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B), at a flow rate of 0.3 mL min-1 in gradient elution. The chromatography was run for 10 min, and analytical curves were built in acetonitrile, plasma, and brain. The PA was detected in positive ion mode with multiple reaction monitoring. The method has shown high selectivity, sensitivity, and throughput. The low quantification limits of 162, 178, and 121 ng mL-1 for acetonitrile, brain, and plasma, respectively, indicate a good detectability of the method. The repeatability and precision observed were within the limits recommended in the literature. The accuracy of the method was verified through high recovery rates (106-118%). The validated method was successfully applied to the pharmacokinetic study of the metabolite PA after the intranasal administration of free or POH-loaded nanoemulsion in rats. The results showed that chitosan nanoemulsion improved the plasma and brain bioavailability of POH, representing a promising alternative to free POH treatment.

Optimized Chitosan-Coated Gliadin Nanoparticles Improved the Hesperidin Cytotoxicity over Tumor Cells

Abstract Hesperidin is a natural compound which is found in citric fruits and presents antitumor and antimicrobial activities. However, the in vivo efficacy of Hesperidin is reduced due to its low oral bioavailability. Protein-based nanoparticles have been applied to improve biological parameters of drugs and natural compounds. Gliadin is a monomeric protein present in wheat. In this study, gliadin-based nanoparticles containing hesperidin were obtained by desolvation technique and a Taguchi orthogonal array design was employed to optimize the formulation. The independent variables were set as concentration of CaCl2 (0.5; 1 or 2%) and stabilizing agent (Pluronic F68, Tween 80 or sodium caseinate). The dependent variables consisted of mean diameter, polydispersity index, zeta potential, and encapsulation efficiency. The results showed significant effects on the dependent variables when 1% CaCl2 and Pluronic F68 were used. The optimized formulation was coated with chitosan to increase the physical stability of the nanoparticles. The final nanoparticles presented a mean diameter of 321 nm and polydispersity index of 0.217, and spherical shape. After coating, the Zeta potential was +21 mV, and the encapsulation efficiency was 73 %. The in vitro release assay showed that about 98% of the drug was released from the nanoparticles after 48 h. Moreover, the nanoparticles reduced hesperidin cytotoxicity on healthy cells (Vero cells) and improved the cytotoxicity on tumor cells (HeLa, PC-3 and Caco-2 cells). Results showed that the chitosan-coated gliadin nanoparticles are potential carriers for hesperidin delivery for cancer treatment.

Bovine serum albumin-based nanoparticles containing the flavonoid rutin produced by nano spray drying

Rutin is a flavonoid compound obtained from different vegetables and fruits; specifically, it is found in the seeds of buckwheat and in fruit peels, particularly citrus. It is also an important constituent of red wine. Rutin exhibits various biological properties including antiviral, vasoprotective, anti-inflammatory, and anticarcinogenic activities. However, its antioxidant activity is the most well studied. Despite the potential for in vitro applications, rutin presents low oral bioavailability that affects its biological activities. Nanoparticles composed of polymers, protein, or lipids are of great importance in the pharmaceutical and nutraceutical areas due to their physicochemical properties, which improve the pharmacokinetics of the drug which is loaded within. This study presents the production of bovine serum albumin (BSA) nanoparticles containing rutin by nano spray drying. Nanoparticles were characterized in terms of mean particle size, size distribution, morphology, zeta potential, and drug content; as well as their antioxidant activity. The optimized spray-drying conditions produced spherical particles with a mean size of 316 nm, zeta potential of −32 mV, and encapsulation efficiency around 32%. Moreover, when antioxidant activity toward the ABTS+ radical was assayed, nanoencapsulation increased the IC50 of rutin by 2-fold. The nano spray-drying process proved to be suitable for the production of rutin-loaded BSA nanoparticles with potential antioxidant activity.

Zein-casein-lysine multicomposite nanoparticles are effective in modulate the intestinal permeability of ferulic acid.

The objective of this study was to develop zein-casein-lysine nanoparticles to modulate the intestinal permeability of ferulic acid (FA), a bioactive compound with proven antioxidant properties. The nanoparticles were obtained by a liquid-liquid dispersion method and were characterized in terms of mean size, polydispersity index, zeta potential, association efficiency (AE), in vitro drug release, x-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR). The in vitro intestinal permeability of nanoparticles was evaluated through Caco-2 and Caco-2/HT29-MTX monoculture and co-culture models, respectively. Nanoparticles presented a mean size of 199 nm and zeta potential of -26 mV. The AE of FA was 23% evaluated by high-performance liquid chromatography (HPLC). XRD showed amorphization of FA after association and FT-IR showed no changes in chemical structures of the compounds after nanoencapsulation. The cytotoxicity assays demonstrated that multicomposite nanoparticles presented a safe profile against Caco-2 and HT29-MTX cells. In the in vitro permeability assay, free FA exhibited higher permeability compared to FA-loaded nanoparticles, possibly due to prolonged FA release from nanoparticles. These new developed zein-casein-lysine nanoparticles may be used for FA sustained delivery by the oral route.

Effects of Silver Nanoparticle Exposure to the Testicular Antioxidant System during the Prepubertal Rat Stage.

Humans and environments are constantly exposed to a wide range of commercial products containing silver nanoparticles (AgNPs) in their composition. The hypothalamic-pituitary-testicular (HP-testicular) axis is sensitive to low doses of AgNPs with repercussions in sperm functionality. The oxidative stress may be related to the pathogenesis of sperm alterations because Ag+ ions are released from AgNPs in the corporal fluids. This study aimed to investigate the effects of AgNP exposure in the antioxidant defense system. For this, the transcript expression and the activity of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX), and glutathione reductase (GSR) enzymes were evaluated in the testis of rats exposed during the prepubertal period to increasing doses of AgNPs (1.875, 3.75, 7.5, or 15 µg of AgNPs/kg). The higher dose of AgNPs (15 µg/kg) investigated promoted increases in the activity of CAT, GPX, and GSR enzymes and in the expression of Gpx4 var1 transcript. The exposure to 7.5 µg/kg of AgNP increased the Gpx4 var1 mRNA expression. In the group that received 3.75 µg of AgNP/kg, the expression of Sod1, Gpx4 var2, and Gsr transcripts was decreased while the Gpx4 var1 mRNA expression was augmented. The lower dose of AgNPs tested (1.875 µg/kg) increased the expression of Cat and Gpx4 var1 transcripts. Thus, AgNP alters the expression and activity of the antioxidant enzymes in a nonmonotonic dose-response curve and directly or indirectly modulates the events related to spermatogenesis process.

Chitosan functionalized poly (ε-caprolactone) nanoparticles for amphotericin B delivery.

Amphotericin B (AmB) is a broad-spectrum antifungal drug used in the treatment of fungal invasive infections. However, its clinical use has been limited due to its side effects and toxicity, especially the nephrotoxicity. Furthermore, AmB presents low aqueous solubility, low permeability over the membranes and poor stability in the gastric environment, which makes it unavailable to be administered by the oral route. In this study, chitosan-coated poly (ε-caprolactone) nanoparticles were developed to provide the oral delivery of AmB and reduce its toxicity. Nanoparticles were obtained by nanoprecipitation and parameters as particle size, polydispersity index (PDI), zeta potential, morphology, in vitro AmB release (in physiological pH and simulated gastrointestinal fluids), state of molecular aggregation, cytotoxicity over erythrocytes and Vero cells line and in vitro antifungal activity were fully investigated. Nanoparticles presented mean size of 318 ± 35 nm, PDI of 0.24 ± 0.02, zeta potential of +36.2 ± 1.8 mV due to chitosan-coating, and 69% of AmB encapsulation. The kinetic release profile of AmB from nanoparticles was of second order and diffusion-governed in pH 7.4. The release in the gastrointestinal simulated fluids showed that the chitosan-coated PCL nanoparticles presented good stability during the time evaluated. AmB was released from nanoparticles in a state of low molecular aggregation. Cytotoxicity over erythrocytes and Vero cells line revealed that nanoencapsulation significantly reduced the AmB-related cytotoxicity (p < 0.05) compared to the free drug. In the antifungal activity against Candida parapsilosis strain, the MIC of AmB-loaded nanoparticles was 5-fold higher than free AmB, but the strain was susceptible to nanostructured AmB. Chitosan-functionalized PCL are potential carriers for the oral AmB delivery, reducing its cytotoxicity and maintaining its activity.

Bovine serum albumin nanoparticles improve the antitumour activity of curcumin in a murine melanoma model.

Curcumin is a natural compound presenting important antitumour activity. However, due to its low aqueous solubility, instability at physiological pH, and low oral bioavailability, its clinical use is limited. Bovine serum albumin (BSA) nanoparticles have been used as drug carriers to improve the drug properties. In this work, curcumin-loaded BSA nanoparticles were developed and the in vitro cytotoxicity over murine melanoma cells and the in vivo antitumour activity in a murine melanoma model were assessed. Nanoparticles presented 150 nm, polydispersity index of 0.16, negative zeta potential, and 45% of curcumin encapsulation efficiency. Curcumin release from nanoparticles was slow and diffusion dependent. In the cytotoxicity assay, free curcumin was more efficient than curcumin-loaded nanoparticles, probably due to the prolonged curcumin release from nanoparticles. However, in a murine melanoma model, curcumin-loaded nanoparticles presented higher antitumour efficiency than free curcumin. BSA nanoparticles are efficient curcumin carriers that may have relevant applications in melanoma treatment.

Mucoadhesive chitosan-coated PLGA nanoparticles for oral delivery of ferulic acid.

This paper describes the development and in vitro evaluation of poly(lactic-co-glycolic acid) (PLGA) nanoparticles coated with chitosan (CS) for oral delivery of ferulic acid (FA). Nanoparticles were obtained by an emulsion evaporation technique and characterized. Furthermore, we evaluated the scavenging activity over hypochlorous acid (HOCl), the cytotoxicity over tumour cells and the in vitro intestinal permeability. Nanoparticles were spherical with a mean diameter of 242 nm, positive zeta potential and 50% of encapsulation efficiency. The in vitro release in phosphate buffered saline (PBS) (pH 7.4) demonstrated a prolonged and biphasic profile diffusion-controlled. In simulated gastrointestinal fluids, about 15% of FA was released in gastric fluid and a negligible release was observed in the intestinal fluid. In the HOCl scavenging activity and cytotoxicity over B16-F10 and HeLa cells, FA-loaded nanoparticles presented the same efficacy of the free drug. Besides, in the antioxidant and cytotoxic assay, CS contributed to FA effects. In the intestinal permeability study, FA-loaded nanoparticles exhibited a permeation of 6% through the Caco-2 monolayer and 20% through the Caco-2/HT29-MTX/Raji B co-culture. CS-coated PLGA nanoparticles are promising carriers for oral delivery of FA.

Preparation and In vitro Evaluation of Efficacy and Toxicity of Polysorbate 80-coated Bovine Serum Albumin Nanoparticles containing Amphotericin B.

OBJECTIVE: In this study, bovine serum albumin (BSA) nanoparticles coated with polysorbate- 80 (PS-80) and containing amphotericin B (AmB) were developed using a coacervation method. METHODS: The nanoparticles were spherical, had a uniform size distribution (polydispersity < 0.25), a small mean size (185 ± 5 nm), a high zeta potential (-38.0 ± 0.7 mV), and a high AmB encapsulation efficiency (93 ± 1%). The AmB release profile was prolonged and diffusion-controlled, resulting in a low degree of AmB aggregation in solution. The physicochemical characteristics of these AmB containing nanoparticles were evaluated by X-ray diffraction, Fourier transform infrared spectroscopy, differential scanning calorimetry, and derivative thermogravimetry and showed that the nanoencapsulation process lead to AmB amorphization while maintaining its chemical integrity. RESULTS: In a hemolysis assay, AmB-loaded PS-80-coated BSA nanoparticles demonstrated an absence of cytotoxicity toward erythrocytes, whereas pure and commercial AmB were highly hemolytic. CONCLUSION: In an assay to assess antifungal activity against Cryptococcus neoformans, AmB-charged PS-80-coated BSA nanoparticles were effective, however, due to the prolonged AmB release from the nanoparticles, the MIC was higher than for pure or commercial AmB. PS-80-coated BSA nanoparticles are potential carriers for the delivery of AmB for the treatment of Cryptococcus sp infections.

Antifungal Activity of Chitosan-Coated Poly(lactic-co-glycolic) Acid Nanoparticles Containing Amphotericin B.

Amphotericin B (AmB) is one of the most used drugs for the treatment of systemic fungal infections; however, the treatment causes several toxic manifestations, including nephrotoxicity and hemolytic anemia. Chitosan-coated poly(lactide-co-glycolide) (PLGA) nanoparticles containing AmB were developed with the aim to decrease AmB toxicity and propose the oral route for AmB delivery. In this work, the antifungal efficacy of chitosan-coated PLGA nanoparticles containing AmB was evaluated in 20 strains of fungus isolates from patients with vulvovaginal candidiasis (01 Candida glabrata and 03 Candida albicans), bloodstream infections (04 C. albicans and 01 C. tropicalis) and patients with urinary tract infection (04 Candida albicans, 02 Trichosporon asahii, 01 C. guilhermondii, 03 C. glabrata) and 01 Candida albicans ATCC 90028. Moreover, the cytotoxicity over erythrocytes was evaluated. The single-emulsion solvent evaporation method was suitable for obtaining chitosan-coated PGLA nanoparticles containing AmB. Nanoparticles were spherical in shape, presented mean particle size about 460 nm, positive zeta potential and encapsulation efficiency of 42%. Moreover, nanoparticles prolonged the AmB release. All the strains were susceptible to plain AmB and nanostructured AmB, according to EUCAST breakpoint version 8.1 (resistant > 1 µg/mL), using broth microdilution method. In C. albicans (urine, blood, and vulvovaginal secretion isolates, and 1 ATCC), the MIC value of AmB-loaded nanoparticles varied from 0.25 to 0.5 µg/mL and EUCAST varied from 0.03 to 0.5 µg/mL. In urine and vulvovaginal secretion isolates of C. glabrata, the MIC value of AmB-loaded nanoparticles varied from 0.25 to 0.5 µg/mL and EUCAST varied from 0.03 to 0.015 µg/mL. In urine isolates of C. guilhermondii, the MIC value of AmB-loaded nanoparticles was 0.12 µg/mL and EUCAST was 0.06 µg/mL. In blood isolates of C. tropicalis, the MIC value of AmB-loaded nanoparticles was 0.5 µg/mL and EUCAST was 0.25 µg/mL. Finally, in urine isolates of T asahii, the MIC value of AmB-loaded nanoparticles was 1 µg/mL and EUCAST varied from 0.5 to 1 µg/mL. In the cytotoxicity assay, plain AmB was highly hemolytic (100% in 24 h) while AmB-loaded chitosan/PLGA nanoparticles presented negligible hemolysis.

PLGA Nanoparticles and Polysorbate-80-Coated PLGA Nanoparticles Increase the In vitro Antioxidant Activity of Melatonin.

BACKGROUND: Melatonin is an endogen substance with several physiological functions, acting as an important antioxidant. Our objective was to prepare nanoparticles composed of poly(lactic-coglycolic acid) (PLGA) coated with polysorbate 80 (PLGA-PS80) or uncoated (PLGA) nanoparticles containing melatonin (MLT) and evaluate their in vitro cytotoxicity over erythrocytes and in vitro antioxidant activity. METHODS: Nanoparticles were obtained by an emulsion-solvent evaporation method and characterized by size, morphology, polydispersity index, zeta potential, encapsulation efficiency, thermal properties and in vitro drug release profile. The in vitro cytotoxicity over erythrocytes was assessed by hemolysis assay and in vitro antioxidant was carried out by colorimetric assay using the radical 2,2-azinobis (3- ethylbenzothiazoline-6-sulfonic acid) diammonium salt) (ABTS·+). RESULTS: Mean size of the PLGA-PS80 and PLGA nanoparticles was 212 and 187 nm, and the encapsulation efficiency of MLT was 26 and 41%, respectively. Nanoparticles were spherical in shape and presented negative zeta potential. MLT was released from nanoparticles following the second order model and PLGA-PS80 presented more prolonged MLT release. CONCLUSION: Cytotoxicity over erythrocytes was assessed and both nanoparticles containing MLT demonstrated lack of hemolysis. Scavenging activity over ABTS·+ demonstrated both nanoparticles containing MLT were more efficient than free drug, and MLT-loaded PLGA nanoparticles presented the higher in vitro antioxidant activity. The study concluded that PLGA and PLGA-PS80 nanoparticles are promising carriers for MLT.

A stability-indicating high performance liquid chromatography method to determine apocynin in nanoparticles.

In this study, we developed and validated a fast, specific, sensitive, precise and stability-indicating high performance liquid chromatography (HPLC) method to determine the drug apocynin in bovine serum albumin (BSA) nanoparticles. Chromatographic analyses were performed on an RP C18 column and using a photodiode array detector at a wavelength of 276 nm. Mobile phase consisted of a mixture of acetonitrile and 1% acetic acid (60:40, v/v), and it was eluted isocratically at a flow rate of 0.8 mL/min. The retention time of apocynin chromatographic peak was 1.65 min. The method was linear, precise, accurate and specific in the range of 5-100 µg/mL. The intra- and inter-day precisions presented relative standard deviation (RSD) values lower than 2%. The method was robust regarding changes in mobile phase proportion, but not for flow rate. Limits of detection and quantitation were 78 ng/mL and 238 ng/mL, respectively. Apocynin was exposed to acid and alkali hydrolysis, oxidation and visible light. The drug suffered mild degradation under acid and oxidation conditions and great degradation under alkali conditions. Light exposure did not degrade the drug. The method was successfully applied to determine the encapsulation efficiency of apocynin in BSA nanoparticles.

Application of a validated HPLC-PDA method for the determination of melatonin content and its release from poly(lactic acid) nanoparticles.

Melatonin is a natural hormone and with the advancement of age its production declines and thereby may result in some neurological disorders. Exogenous administration of melatonin has been suggested as a neuroprotective agent. Due to its low oral bioavailability, the loading of melatonin in polymeric nanoparticles could be an important tool to effectively use exogenous melatonin. The quantification of the incorporated drug within polymeric nanoparticles is an important step in nanoparticles characterization. An analytical method using high performance liquid chromatography equipped with photodiode array detector (HPLC-PDA) was developed and validated for melatonin determination in poly (lactic acid) nanoparticles obtained by a single emulsion-solvent evaporation technique. The melatonin in vitro release profile also was determined by the HPLC method. Mobile phase consisted of acetonitrile: water (65:35, v/v) pumped at a flow rate of 0.9 mL/min, in the isocratic mode and PDA detector was set at 220 nm. The method was validated in terms of the selectivity, linearity, precision, accuracy, robustness, limits of detection and quantification. Analytical curve was linear over the concentration range of 10-100 µg/mL, and limits of detection and quantification were 25.9 ng/mL and 78.7 ng/mL, respectively. The mean recovery for melatonin was 100.47% (RSD = 1.25%, n = 9). In the intra- and inter-assay, the coefficient of variation was less than 2%. Robustness was proved performing changes in mobile phase, column temperature and flow rate. The method was suitable for the determination of melatonin encapsulation efficiency in poly(lactic acid) nanoparticles and for the evaluation of melatonin in vitro release profile.