Establishment and characterization of an in vitro 3D ovarian cancer model for drug screening assays.

The acquired drug chemoresistance represents the main challenge of the ovarian cancer treatment. In addition, the absence of an adequate in vitro model able to reproduce the native tumor environment can contribute to the poor success rate of pre-clinical studies of new compounds. Three-dimensional (3D) culture models have been recently used for drug screening purposes due to their ability to reproduce the main characteristics of in vivo solid tumors. Here we describe the establishment and characterization of 3D ovarian cancer spheroids using different adenocarcinoma tumor cell lines (SKOV-3 and OVCAR-3 cells) in two different 3D scaffold-free methods: forced-floating in ultra-low attachment (ULA) plates and hanging drop (HD). Spheroids were evaluated in both 3D cultures in order to establish the best condition to perform the drug response analysis with Paclitaxel, a common drug used to treat ovarian cancer. SKOV-3 and OVCAR-3 spheroids with the desired characteristics (roundness close to 1.0 and diameter in the 200-500 µm range) were obtained using both methods after addition of the methylcellulose (MC) in the culture medium (0.25% and 0.5%, w/v). We also observed the presence of microvilli on the surface of the spheroids, higher presence of apoptotic cells and higher drug resistance, when compared with 2D cultures. The 3D cultures obtained seem to provide more reliable results in terms of drug response than those provided by 2D monolayer culture. The forced floating method was considered more suitable and straightforward to generate ovarian cancer spheroids for drug screening/cytotoxicity assays.

Skin permeation, biocompatibility and antitumor effect of chloroaluminum phthalocyanine associated to oleic acid in lipid nanoparticles.

The objective of this study was to develop and characterize lipid nanoparticles (LNs) containing chloroaluminum phthalocyanine (ClAlPc) to reduce the aggregation of the drug and improve its skin penetration and its antitumor effect. LNs were prepared and characterized by using stearic acid (SA) as solid lipid and oleic acid (OA) as liquid lipid in different proportions. in vitro and in vivo skin penetration was evaluated using modified Franz diffusion cells and fluorescence microscopy, respectively. in vitro biocompatibility and Photodynamic Therapy (PDT) were performed using L929-fibroblasts cell line and A549 cancer cell line and melanoma BF16-F10, respectively. OA promoted the increase in the encapsulation efficiency and drug loading, reaching values of 95.8% and 4%, respectively. The formulation with 40% OA (NLC 40) showed a significantly higher (p < 0.01) amount of drug retained in the skin compared to other formulations. All formulations developed were considered biocompatible. PDT evidenced the antitumor efficacy of NLC 40 with reduced cell viability for approximately 10% of cancer cells, demonstrating that the presence of OA in the NLC seems to potentialize this antitumor effect. PDT in BF16-F10 melanoma using NLC 40 resulted in a reduction in mean cell viability of approximately 99%. According to the results obtained, the systems developed may be promising for the incorporation of ClAlPc in the treatment of skin cancer by photodynamic therapy.

Targeted lipid nanoparticles for antisense oligonucleotide delivery.

Antisense oligonucleotides (AS-ODNs) are short, single-stranded DNA molecules designed to bind specifically to a target messenger RNA (mRNA) and down-regulate gene expression. Despite being a promising class of therapeutics for a variety of diseases, they face major hurdles limiting their clinical application, including low intracellular delivery and poor in vivo stability. Among strategies available to enhance delivery, lipid nanoparticles have gained considerable attention. Active targeting of carriers of AS-ODNs is likely to further enhance delivery efficiency. For that, ligands for overexpressed receptors on the cell surface can be linked to the lipid nanoparticle, facilitating intracellular uptake, resulting in improved efficacy and reduced systemic toxicity. These include cell penetrating peptides (CPPs), transferrin, folate, oligosaccharides, polysaccharides and antibodies. Although targeted-lipid nanoparticles have been shown to enhance intracellular delivery and therapeutic effect of AS-ODNs, no clinical evaluation has been conducted yet. Therefore, more efforts are needed to turn these promising tools into clinical products.

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 µ 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.

Assessment of the mutagenic activity of extracts of brazilian propolis in topical pharmaceutical formulations on Mammalian cells in vitro and in vivo.

Propolis possesses various biological activities such as antibacterial, antifungal, anti-inflammatory, anesthetic and antioxidant properties. A topically applied product based on Brazilian green propolis was developed for the treatment of burns. For such substance to be used more safely in future clinical applications, the present study evaluated the mutagenic potential of topical formulations supplemented with green propolis extract (1.2, 2.4 and 3.6%) based on the analysis of chromosomal aberrations and of micronuclei. In the in vitro studies, 3-h pulse (G(1) phase of the cell cycle) and continuous (20 h) treatments were performed. In the in vivo assessment, the animals were injured on the back and then submitted to acute (24 h), subacute (7 days) and subchronic (30 days) treatments consisting of daily dermal applications of gels containing different concentrations of propolis. Similar frequencies of chromosomal aberrations were observed for cultures submitted to 3-h pulse and continuous treatment with gels containing different propolis concentrations and cultures not submitted to any treatment. However, in the continuous treatment cultures treated with the 3.6% propolis gel presented significantly lower mitotic indices than the negative control. No statistically significant differences in the frequencies of micronuclei were observed between animals treated with gels containing different concentrations of propolis and the negative control for the three treatment times. Under the present conditions, topical formulations containing different concentrations of green propolis used for the treatment of burns showed no mutagenic effect in either test system, but 3.6% propolis gel was found to be cytotoxic in the in vitro test.

Preparation and characterization of D, L-PLA loaded 17-ß-Estradiol valerate by emulsion/evaporation methods.

PLA microparticles containing 17-ß-estradiol valerate were prepared by an emulsion/evaporation method in order to sustain drug release. This system was characterized concerning particle size, particle morphology and the influence of formulation and processing parameters on drug encapsulation and in vitro drug release. The biodegradation of the microparticles was observed by tissue histological analysis. Scanning electron microscopy and particle size analysis showed that the microparticles were spherical, presenting non-aggregated homogeneous surface and had diameters in the range of 718-880 nm (inert micro-particles) and 3-4 µm (drug loaded microparticles). The encapsulation efficiency was ∼80%. Hormone released from microparticles was sustained. An in vivo degradation experiment confirmed that microparticles are biodegradable. The preparation method was shown to be suitable, since the morphological characteristics and efficiency yield were satisfactory. Thus, the method of developed microparticles seems to be a promising system for sustained release of 17-ß-estradiol.

Preparation and characterization of chitosan-treated alginate microparticles incorporating all-trans retinoic acid.

Chitosan treated alginate microparticles were prepared with the purpose of incorporating all-trans retinoic acid (ATRA) using an inexpensive, simple and fast method, enhancing dermal localization and sustaining the release of ATRA into the skin. Microparticles characterization, drug-polymer interaction, release profile and in vitro skin retention were investigated. Microparticles presented spherical shape and drug loading capacity of 47%. The drug content of these microparticles was affected by ATRA concentration and by the solvent used and it was more weakly affected by chitosan concentration. The release of ATRA was also affected by chitosan concentration. Microparticles prepared with 0.4% chitosan (w/w) resulted in drug release with a more sustained profile. The results of in vitro retention studies showed that chitosan treated alginate microparticles decreased the drug retention in the stratum corneum (SC), where occur the skin irritation, but maintained the ATRA concentration in the deeper skin layers, where occur the pathologies treated with ATRA. Then, the microparticles developed in this work can be a good candidate to improve the topical therapy with retinoid.

Indocyanine green nanoparticles useful for photomedicine.

OBJECTIVE: The aim of this study was to evaluate the potential application of biodegradable nanoparticles (NPs) containing indocyanine green (ICG) in photodynamic therapy (PDT). METHODS: Important parameters, such as particle size and external morphology, were established by dynamic light scattering (DLS) and scanning electron microscopy (SEM). Also, drug encapsulation efficiency and in vitro release behavior were evaluated by spectroscopic methods. RESULTS: The particles are spherical in shape, they exhibit an 817-nm diameter, and they have a low tendency to aggregate. The loading efficiency was 65%. ICG photophysical parameters showed a bathocromic shift in ICG-loaded nanoparticles (ICG-NP). Analysis of the cell P388-D1 in the presence of the ICG-NP by SEM showed that the majority of the nanoparticles were uptaken by phagocytic cells after 2 h of incubation. After laser irradiation photodamage was observed in P388-D1 cells where ICG-NPs had been uptaken by phagocytic cells. CONCLUSION: Polymeric NPs work as an efficient drug delivery system for PDT drugs, and this approach can be used in the administration of amphiphilic photosensitizers in the treatment of neoplasic cells.

Preparation, characterization, photocytotoxicity assay of PLGA nanoparticles containing zinc (II) phthalocyanine for photodynamic therapy use.

Nanoparticles containing Zinc (II) Phthalocyanine (ZnPc) were prepared by a spontaneous emulsification diffusion method utilizing poly-(D,L lactic-co-glycolic acid) (PLGA), characterized and available in cellular culture. The process yield and encapsulation efficiency were 60% and 80%, respectively. The nanoparticles have a mean diameter of 200 nm, a narrow size distribution with polydispersive index of 0.15, smooth surface and spherical shape. ZnPc loaded nanoparticles maintain their photophysical behaviour after the encapsulation process. Photosensitizer released from nanoparticles was sustained with a burst effect of 10% for 3 days. The photocytotoxicity was evaluated on P388-D1 cells. They were incubated with ZnPc loaded Np by 6 h and exposed to light (675 nm) for 120 s, and light dose of 30 J cm-2. After 24 h of incubation, the cellular viability was determined, obtaining 60% of cellular death. All the physical-chemical and photobiological measurements performed allowed one conclude that ZnPc loaded PLGA nanoparticles are a promising drug delivery system for PDT.

Photobiological and ultrastructural studies of nanoparticles of poly(lactic-co-glycolic acid)-containing bacteriochlorophyll-a as a photosensitizer useful for PDT treatment.

The interaction of polymeric nanoparticles formulated from the biodegradable polymer poly(DL-lactide-co-glycolide) loaded with bacteriochlorophyll-a was studied in homogeneous solution and in vitro in the presence of a macrophage cell line (P388-D1-ATCC). Photodynamic therapy (PDT) activity after different laser doses also was investigated. Scanning electron microscopy analysis of cell phagocyte nanoparticles showed that after 30 min of incubation most of the nanoparticles are in a clear adhesion process to the cell surface. The majority of nanoparticles became phagocytic after 2 hr of incubation time. After laser irradiation of the dye-containing system a total photodamage by nanoparticle phagocyte cells was observed and the cell survival was quantified by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test. Our results indicate that polymeric nanoparticles work as an efficient drug delivery system for PDT drugs. This approach can be widely used for many other hydrophobic photosensitizers with higher aggregation tendency in neoplastic cell treatment.