In Vitro Antifungal Activity and Toxicity of Itraconazole in DMSA-PLGA Nanoparticles

Itraconazole (ITZ) is a drug used to treat various fungal infections and may cause side effects. The aim of this study was to develop and evaluate the in vitro activity of DMSA-PLGA nanoparticles loaded with ITZ against Paracoccidioides brasiliensis, as well as their cytotoxicity. Nanoparticles were prepared using the emulsification-evaporation technique and characterized by their encapsulation efficiency, morphology (TEM), size (Nanosight) and charge (zeta potential). Antifungal efficacy in P brasiliensis was determined by minimal inhibition concentration (MIC), and cytotoxicity using MU assay. ITZ was effectively incorporated in the PLGA-DMSA nanoparticles with a loading efficiency of 72.8 +/- 3.50%. The shape was round with a solid polymeric structure, and a size distribution of 174 +/- 86 nm (Average +/- SD). The particles were negatively charged. ITZ-NANO presented antifungal inhibition (MIC = 6.25 ug/mL) against P brasiliensis and showed lower in vitro cytotoxicity than free drug (ITZ).

Synthetic long peptide-based vaccine formulations for induction of cell mediated immunity: A comparative study of cationic liposomes and PLGA nanoparticles.

Nanoparticulate formulations for synthetic long peptide (SLP)-cancer vaccines as alternative to clinically used Montanide ISA 51- and squalene-based emulsions are investigated in this study. SLPs were loaded into TLR ligand-adjuvanted cationic liposomes and PLGA nanoparticles (NPs) to potentially induce cell-mediated immune responses. The liposomal and PLGA NP formulations were successfully loaded with up to four different compounds and were able to enhance antigen uptake by dendritic cells (DCs) and subsequent activation of T cells in vitro. Subcutaneous vaccination of mice with the different formulations showed that the SLP-loaded cationic liposomes were the most efficient for the induction of functional antigen-T cells in vivo, followed by PLGA NPs which were as potent as or even more than the Montanide and squalene emulsions. Moreover, after transfer of antigen-specific target cells in immunized mice, liposomes induced the highest in vivo killing capacity. These findings, considering also the inadequate safety profile of the currently clinically used adjuvant Montanide ISA-51, make these two particulate, biodegradable delivery systems promising candidates as delivery platforms for SLP-based immunotherapy of cancer.

Development of praziquantel-loaded PLGA nanoparticles and evaluation of intestinal permeation by the everted gut Sac model

Praziquantel has been shown to be highly effective against all known species of Schistosoma infecting humans. Spherical nanoparticles made of poly(D,L-lactide-co-glycolide) acid with controlled size were designed as drug carriers. Praziquantel, a hydrophobic drug, was entrapped into the polymeric nanoparticles with 30% (w/w) of theoretical loading. The nanoparticles size was approximately of 350 nm with 66% of encapsulation efficiency. The everted gut sac model shows to be efficient to evaluate the drug permeation through the intestinal membrane. The results show that free praziquantel presents 4-fold times more permeation than praziquantel-loaded PLGA nanoparticles and physical mixture. For this drug, in special, this result can be interesting, since the nanoparticulate system can behave as a drug reservoir and/or to have a more localized effect in intestinal membrane for a prolonged period of time, since great amounts of parasites can be usually found in the mesenteric veins.

Praziquantel-loaded PLGA nanoparticles: preparation and characterization

Nanoparticles containing praziquantel made of Poly (D,L-lactide-co-glycolide) were designed by an emulsion-solvent evaporation method. Two organic solvents were separately utilized as disperse phase: methylene chloride and ethyl acetate. The size of the particles prepared with the former solvent was bigger than the particles prepared with the latter. The entrapment efficiency was bigger when methylene chloride was used, 79.82% in comparison with 29.27% by using ethyl acetate. DSC and infrared studies showed that no strong chemical interaction between drug and polymer occurred. Release kinetics of praziquantel, used as a model drug, was governed not only by actual drug loading but also by particles size. The higher the drug content and the smaller the particle size resulted in faster drug release.

PLGA nanoparticles containing praziquantel: effect of formulation variables on size distribution

Praziquantel has been shown to be highly effective against all known species of Schistosoma infecting humans. Spherical nanoparticulate drug carriers made of poly(D,L-lactide-co-glycolide) acid with controlled size were designed. Praziquantel, a hydrophobic molecule, was entrapped into the nanoparticles with theoretical loading varying from 10 to 30% (w/w). This investigates the effects of some process variables on the size distribution of nanoparticles prepared by emulsion-solvent evaporation method. The results show that sonication time, PLGA and drug amounts, PVA concentration, ratio between aqueous and organic phases, and the method of solvent evaporation have a significant influence on size distribution of the nanoparticles. (C) 2004 Elsevier B.V. All rights reserved.

Thermoanalytical study of praziquantel-loaded PLGA nanoparticles

Polymeric nanoparticles have received great attention as potential controlled drug delivery systems. Biodegradable polymers has been extensively used in the development of these drug carriers, and the polyesters such as polylactic acid, polyglycolic acid and their copolymers as poly-lactide-co- glycolide are the most used, considering its biocompatibility and biodegradability. Thermal analysis techniques have been used for pharmaceutical substances for more than 30 years and are routine methods for screening drug-excipient interactions. The aim of this work is to use thermal analysis to characterize PLGA nanoparticles containing a hydrophobic drug, praziquantel. The results show that the drug is in an amorphous state or in disordered crystalline phase of molecular dispersion in the PLGA polymeric matrix and that the microencapsulation process did not interfere with the chemical structure of the polymer, mantaining the structural drug integrity.

Antioxidant Effects of Quercetin and Catechin Encapsulated into PLGA Nanoparticles

Polymeric nanoparticles (PLGA) have been developed for the encapsulation and controlled release of quercetin and catechin. Nanoparticles were fabricated using a solvent displacementmethod. Physicochemical properties were measured by light scattering, scanning electron microscopy and zeta-potential, X-ray diffraction, infrared spectroscopy and differential scanning calorimetry. Encapsulation efficiency and in vitro release profiles were obtained from differential pulse voltammetry experiments. Antioxidant properties of free and encapsulated flavonoids were determined by TBARS, fluorescence spectroscopy and standard chelating activity methods. Relatively small (d approximate to 400 nm) polymeric nanoparticles were obtained containing quercetin or catechin in a non-crystalline form (EE approximate to 79%) and the main interactions between the polymer and each flavonoid were found to consist of hydrogen bonds. In vitro release profiles were pH-dependant, the more acidic pH, the faster release of each flavonoid from the polymeric nanoparticles. The inhibition of the action of free radicals and chelating properties, were also enhanced when quercetin and catechin were encapsulated within PLGA nanoparticles. The information obtained from this study will facilitate the design and fabrication of polymeric nanoparticles as possible oral delivery systems for encapsulation, protection and controlled release of flavonoids aimed to prevent oxidative stress in human body or food products.

Curcumin-loaded into PLGA nanoparticles

The incorporation of the curcumin into poly(lactic-co-glycolic)acid (PLGA) nanospheres by the nanoprecipitation technique, the characterization of the nanoparticles and the schistosomicidal activity of the curcumin-loaded into PLGA nanospheres were reported. The incorporation process occurred with high efficiency and the images of field-emission scanning electron microscopy (FESEM) revealed the production of spherically shaped particles. According to the dynamic light scattering measurements, the particles are nanometric and monodisperse. The curcumin-loaded PLGA nanoparticles (50 and 100 mu M) caused the death of all worms and a separation between 50% and 100% of Schistosoma mansoni couples at concentrations from 30 mu M. Moreover, the curcumin-loaded PLGA nanoparticles also decreased the motor activity and caused partial alterations in the tegument of adult worms. This study marks the first time that schistosomicidal activity has been reported for curcumin-loaded PLGA nanoparticles.

Cationic Bovine Serum Albumin (CBA) conjugated Poly Lactic-co-Glycolic Acid (PLGA) nanoparticles for extended delivery of methotrexate into brain tumor

Current treatment strategies for the treatment of brain tumor have been hindered primarily by the presence of highly lipophilic insurmountable blood-brain barrier (BBB). The purpose of current research was to investigate the efficiency of engineered biocompatible polymeric nanoparticles (NPs) as drug delivery vehicle to bypass the BBB and enhance biopharmaceutical attributes of anti-metabolite methotrexate (MTX) encapsulated NPs. The NPs were prepared by solvent diffusion method using cationic bovine serum albumin (CBA), and characterized for physicochemical parameters such as particle size, polydispersity index, and zeta-potential; while the surface modification was confirmed by FTIR, and NMR spectroscopy. Developed NPs exhibited zestful relocation of FITC tagged NPs across BBB in albino rats. Further, hemolytic studies confirmed them to be non-toxic and biocompatible as compared to free MTX. In vitro cytotoxicity assay of our engineered NPs on HNGC1 tumor cells proved superior uptake in tumor cells; and elicited potent cytotoxic effect as compared to plain NPs and free MTX solution. The outcomes of the study evidently indicate the prospective of CBA conjugated poly (D,L-lactide-co-glycolide) (PLGA) NPs loaded with MTX in brain cancer bomber with amplified capability to circumvent BBB.

Effects of ball-milling on PLGA polymer and its implication on lansoprazole-loaded nanoparticles

PLGA is a biodegradable polymer utilised widely in pharmaceutical research for the encapsulation of a wide range of drugs as nano particulate systems. This study investigates the impact of rotary ball milling on the physical properties of PLGA and its influence on nanoparticle formation prepared using the solvent displacement technique. By applying mechanical stress to the polymer and altering its physical appearance and molecular weight, the loading of lansoprazole within the nanoparticles was increased to 96%, with a reduction in particle size. The results indicate that rotary ball milling significantly reduces particle size, increases lansoprazole loading and improves the release profile for lansoprazole loaded PLGA nanoparticles.

Nanoparticles for gene delivery to retinal pigment epithelial cells.

PURPOSE: To evaluate the safety and potential use of poly(lactic) acid (PLA) and poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) as vectors for gene transfer to RPE cells. METHODS: Experiments were conducted with primary bovine RPE cells and with the ARPE-19 human RPE cell line. Rhodamine loaded NPs were used to study factors influencing the internalization process by the various RPE cells: concentrations of NPs, duration of contact time, stage of cell culture and ambient temperature. The extent of NPs internalization was evaluated by fluorescence and phase microscopy. Potential NP toxicity was measured by the trypan blue exclusion dye test and the MTT method. Green fluorescent protein (GFP) plasmid or red nuclear fluorescent protein (RNFP) plasmid were sequestered in NPs. The ability ot these "loaded" NPs to generate gene transfection and protein expression in RPE cells was assessed both in vivo and in vitro by fluorescence and confocal microscopy. RESULTS: The extent of NP internalization in cultured cells increases with their concentration reaching a plateau at 1 mg/ml and a contact time of up to 6 h. Temperature and culture stage did not influence the in vitro internalization process. No toxic effects on RPE cells could be detected when these were incubated with up to 4 mg/ml of NPs. In human and bovine RPE cells incubated with GFP loaded NPs, cytoplasmic green fluorescence was observed in 14+/-1.65% of the cultured cells. Incubation with RNFP loaded NPs yielded a nuclear red fluorescence in 18.9+/-1.6% of the cells. These percentage levels of expression initially detected after 48 h of incubation remained unchanged during the following 8 additional days in culture. No significant differences in the extent of cytoplasm or nuclear fluorescence expression were observed between bovine or human RPE cultured cells. In vivo, a preferential RNFP expression within the RPE cell layer was detected after intra vitreous injection of RNFP plasmid loaded NPs. CONCLUSIONS: The ability of PLGA NPs to sequester plasmids, their nontoxic characteristics, and rapid internalization enables gene transfer and expression in RPE cells. These findings may be of potential use when designing future gene therapy strategies for ocular diseases of the posterior segment.

Poly(lactic acid-glycolic acid) nanoparticles markedly improve immunological protection provided by peptide P10 against murine paracoccidioidomycosis

Background and purpose: The present study reports on the preparation and testing of a sustained delivery system for the immunomodulatory peptide P10 aimed at reducing the in vivo degradation of the peptide and the amount required to elicit a protective immune response against paracoccidioidomycosis. Experimental approach: BALB/c mice were infected with the yeast Paracoccidioides brasiliensis to mimic the chronic form of paracoccidioidomycosis. The animals were treated daily with sulfamethoxazole/trimethoprim alone or combined with peptide P10, either emulsified in Freund`s adjuvant or entrapped in poly(lactic acid-glycolic acid) (PLGA) nanoparticles at different concentrations (1 mu g, 5 mu g, 10 mu g, 20 mu g or 40 mu g center dot 50 mu L-1). Therapeutic efficacy was assessed as fungal burden in tissues and the immune response by quantitative determination of cytokines. Key results: Animals given combined chemotherapy and P10 nanotherapy presented a marked reduction of fungal load in the lungs, compared with the non-treated animals. After 30 days of treatment, P10 entrapped within PLGA (1 mu g center dot 50 mu L-1) was more effective than `free` P10 emulsified in Freund`s adjuvant (20 mu g center dot 50 mu L-1), as an adjuvant to chemotherapy. After treatment for 90 days, the higher doses of P10 entrapped within PLGA (5 or 10 mu g center dot 50 mu L-1) were most effective. Treatment with P10 emulsified in Freund`s adjuvant (20 mu g center dot 50 mu L-1) or P10 entrapped within PLGA (1 mu g center dot 50 mu L-1) were accompanied by high levels of interferon-gamma in lung. Conclusions and implications: Combination of sulfamethoxazole/trimethoprim with the P10 peptide entrapped within PLGA demonstrated increased therapeutic efficacy against paracoccidioidomycosis. P10 incorporation into PLGA nanoparticles dramatically reduced the peptide amount necessary to elicit a protective effect.

Evaluation of nanoparticles loaded with benzopsoralen in rat peritoneal exudate cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Development of docetaxel and alendronate-loaded chitosan-conjugated polylactide-co-glycolide nanoparticles: In vitro characterization in osteosarcoma cells

Purpose: To develop docetaxel (DTX)- and alendronate (ALN)-loaded, chitosan (CS)-conjugated polylactide- co-glycolide (PLGA) nanoparticles (NPs) to increase therapeutic efficacy in osteosarcoma cells. Methods: Drug-loaded PLGA NPs were prepared by nanoprecipitation and chemically conjugated by the carboxylic group of PLGA to the amine-bearing CS polymer. The nanocarrier was characterized by dynamic light scattering, transmission electron microscopy, scanning electron microscopy, and differential scanning calorimetry as well as by in vitro drug release and cell culture studies. Results: NP size was within the tumour targeting range (~200 nm) with an effective positive charge (20 mV), thus increasing cellular uptake efficiency. Morphological analysis revealed clear spherical particles with uniform dispersion. The NPs exhibited identical sustained release kinetics for both DTX and ALN. CS-conjugated PLGA with dual-drug-loaded (DTX and AL) NPs showed typical time-dependent cellular uptake and also displayed superior cytotoxicity in MG-63 cells compared with blank NPs, which were safe and biocompatible. Conclusion: Combined loading of DTX and ALN in NPs increased the therapeutic efficacy of the formulation for osteosarcoma treatment, thus indicating the potential benefit of a combinatorial drug regimen using nanocarriers for effective treatment of osteosarcoma.

Microparticles as a Strategy for Low-Molecular-Weight Heparin Delivery

The aims of this work were preparation and physical-chemical characterization of a microparticulate release system for delivery of enoxaparin sodium (ENX), a low-molecular-weight heparin, as a potential vehicle for optimization of deep venous thrombosis therapy. Microparticles (MPs) containing ENX were prepared from polylactide-co-glycolic acid [PLGA; (50: 50)] by a double emulsification/solvent evaporation method. The preparation parameters, such as proportion ENX/PLGA, surfactant concentration, type, time, and speed of stirring, were evaluated. The encapsulation efficiency and yield process were determined and optimized, and the in vitro release profile was analysed at 35 days. The MPs showed a spherical shape with smooth and regular surfaces. The size distribution showed a unimodal profile with an average size of 2.0 +/- 0.9 mu m. The low encapsulation efficiency (< 30%), characteristic of hydrophilic macromolecules was improved, reaching 50.2% with a procedure yield of 71.3%. The in vitro profile of ENX release from the MPs was evaluated and showed pseudo-zero-order kinetics. This indicated that diffusion was the main drug release mechanism. (C) 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:1783-1792, 2011