Domingos Ferreira

Preparation, characterization and biocompatibility studies on risperidone-loaded solid lipid nanoparticles (SLN): high pressure homogenization versus ultrasound.

The suitability of solid lipid nanoparticles (SLN) for the encapsulation of risperidone (RISP), an antipsychotic lipophilic drug, was assessed for oral administration. The hot high pressure homogenization (HPH) and the ultrasound (US) technique were used as production methods for SLN. All the studies on the SLN formulations were done in parallel, in order to compare the results and conclude about the advantages and limitations of both techniques. The particle sizes were in the nanometer range for all prepared SLN formulations and the zeta potential absolute values were high, predicting good long-term stability. Optical analyses demonstrated the achievement of stable colloidal dispersions. Physicochemical characterization of dispersions and bulk lipids, performed by differential scanning calorimetry (DSC) and X-ray assays, support prediction of occurrence of drug incorporation in the SLN and good long term stability of the systems. The toxicity of SLN with Caco-2 cells and the existence of contaminations derived from the production equipments were assessed by the (4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) assay. The results showed 90% of cell viability after SLN exposure, with no significant differences within all prepared formulations (p > 0.05). From this study, we conclude that SLN can be considered as efficient carriers for RISP encapsulation. Moreover, HPH and US revealed to be both effective methods for SLN production.

Establishment of a triple co-culture in vitro cell models to study intestinal absorption of peptide drugs

In vitro cell culture models for studying oral drug absorption during early stages of drug development have become a useful tool in drug discovery and development, with respect to substance throughput and reproducibility. The aim of this study was to establish an in vitro cellular model based on human colon carcinoma Caco-2, mucus-producing HT29, and Raji B cells in order to design a model that more accurately mimics the small intestinal epithelial layer. Normal oriented model was set up by seeding co-cultures of Caco-2 and HT29 cells into Transwell filters and maintained under identical conditions following addition of Raji B to the basolateral chamber. Inverted model was set up seeding Caco-2 and HT29 cells on the basolateral chamber and then transferred in the Transwell device with the epithelial cells facing the basolateral chamber following Raji B addition to the apical compartment. Morphological differences on size and thickness of cell membranes were detected between the models studied by using fluorescence microscopy. On the triple co-culture models, cell membranes were increasing in size and thickness from the Caco-2 to Caco-2/HT29 and Caco-2/Raji B. Also, the nuclei seem to be larger than in the other studied models. Insulin permeation was higher on the triple co-culture model when compared to the Caco-2/HT29 co-culture model. Also, insulin permeation as mediated by nanoparticles and insulin solution permeation was higher on the normal oriented Caco-2/HT29/Raji B model as compared to the inverted model. Overall, our results suggest that Caco-2/HT29/Raji B triple co-culture normal oriented cellular model may be reliable to obtain a more physiological, functional, and reproducible in vitro model of the intestinal barrier to study protein absorption, both in solution and when delivered by nanocarriers.

Microencapsulation of hemoglobin in chitosan-coated alginate microspheres prepared by emulsification/internal gelation

Chitosan-coated alginate microspheres prepared by emulsification/internal gelation were chosen as carriers for a model protein, hemoglobin (Hb), owing to nontoxicity of the polymers and mild conditions of the method. The influence of process variables related to the emulsification step and microsphere recovering and formulation variables, such as alginate gelation and chitosan coating, on the size distribution and encapsulation efficiency was studied. The effect of microsphere coating as well its drying procedure on the Hb release profile was also evaluated. Chitosan coating was applied by either a continuous microencapsulation procedure or a 2-stage coating process. Microspheres with a mean diameter of less than 30 μm and an encapsulation efficiency above 90% were obtained. Calcium alginate cross-linking was optimized by using an acid/CaCO3 molar ratio of 2.5, and microsphere-recovery with acetate buffer led to higher encapsulation efficiency. Hb release in gastric fluid was minimal for air-dried microspheres. Coating effect revealed a total release of 27% for 2-stage coated wet microspheres, while other formulations showed an Hb release above 50%. Lyophilized microspheres behaved similar to wet microspheres, although a higher total protein release was obtained with 2-stage coating. At pH 6.8, uncoated microspheres dissolved in less than 1 hour; however, Hb release from air-dried microspheres was incomplete. Chitosan coating decreased the release rate of Hb, but an incomplete release was obtained. The 2-stage coated microspheres showed no burst effect, whereas the 1-stage coated microspheres permitted a higher protein release.

Solid lipid nanoparticles as intracellular drug transporters: an investigation of the uptake mechanism and pathway.

The aim of this work was to develop a systematic analysis of the cellular internalisation mechanism and pathway of solid lipid nanoparticles (SLN) internalisation. To evaluate if SLN show cell uptake and to understand the mechanism of internalisation, four human glioma cell lines (A172, U251, U373 and U87) and a human macrophage cell line (THP1) were used. For this purpose rhodamine 123 (R123) was loaded into SLN coated with polysorbate 60 and 80. Fluorescence microscopy and flow cell cytometry techniques were assessed to study internalisation of these systems within the cells. MTT studies were performed to evaluate the cytotoxicity of the R123-loaded SLN. To assess the SLN internalisation mechanism and intracellular pathway, excluding endocytosis mechanisms were applied. Our results revealed that R123-loaded SLN with mean size below 200 nm and slight negative surface charge (around -20 mV) have the ability to be internalised by gliomas in a higher amount than by macrophages. The mechanism of internalisation was found to be mainly through a clathrin-dependent endocytic pathway. In addition, the cytotoxicity of SLN was higher for gliomas than for macrophages. These results suggest that SLN can be a promising alternative in brain tumours treatment.

Physicochemical investigation of the effects of water-soluble polymers on vinpocetine complexation with β-cyclodextrin and its sulfobutyl ether derivative in solution and solid state

The studies reported in this work aimed to elucidate the inclusion complex formation of vinpocetine (VP), a poorly water-soluble base type drug, with beta-cyclodextrin (betaCD) and its sulfobutyl ether derivative (sulfobutyl ether beta-cyclodextrin (SBEbetaCD)), with or without water-soluble polymers (PVP and HPMC), by thoroughly investigating their interactions in solution and solid state. Phase solubility studies were carried out to evaluate the solubilizing power of both cyclodextrins (CDs), in association with water-soluble polymers, towards VP and to determine the apparent stability constants (Kc) of the complexes. SBEbetaCD showed higher solubilizing efficacy toward VP than the parent betaCD due to its greater solubility and complexing abilities, what was reflected in higher Kc values. Improvement in Kc values for ternary complexes clearly proves the benefit on the addition of water-soluble polymers to promote higher complexation efficiency. VP-CDs (1:1) binary and ternary systems were prepared by physical mixing, kneading, co-evaporation, and lyophilization methods. In the solid state, drug-carrier interactions were studied by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), X-ray diffractometry (XRD) and Fourier-transform infrared spectroscopy. The results of these analysis suggested the formation of new solid phases, some of them in amorphous state, allowing to the conclusion of strong evidences of binary and ternary inclusion complex formation between VP, CD and water-soluble polymers, particularly for co-evaporated and lyophilized binary and ternary products.

Nanotechnology and pulmonary delivery to overcome resistance in infectious diseases

Abstract Used since ancient times especially for the local treatment of pulmonary diseases, lungs and airways are a versatile target route for the administration of both local and systemic drugs. Despite the existence of different platforms and devices for the pulmonary administration of drugs, only a few formulations are marketed, partly due to physiological and technological limitations. Respiratory infections represent a significant burden to health systems worldwide mainly due to intrahospital infections that more easily affect immune-compromised patients. Moreover, tuberculosis (TB) is an endemic infectious disease in many developing nations and it has resurged in the developed world associated with the human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) epidemic. Currently, medicine faces the specter of antibiotic resistance. Besides the development of new anti-infectious drugs, the development of innovative and more efficient delivery systems for drugs that went off patent appears as a promising strategy pursued by the pharmaceutical industry to improve the therapeutic outcomes and to prolong the utilities of their intellectual property portfolio. In this context, nanotechnology-based drug delivery systems (nano-DDS) emerged as a promising approach to circumvent the limitations of conventional formulations and to treat drug resistance, opening the hypothesis for new developments in this area.

Brain delivery of camptothecin by means of solid lipid nanoparticles: Formulation design, in vitro and in vivo studies

For the purpose of brain delivery upon intravenous injection, formulations of camptothecin-loaded solid lipid nanoparticles (SLN), prepared by hot high pressure homogenisation, were designed. Incorporation of camptothecin in the hydrophobic and acidic environment of SLN matrix was chosen to stabilise the lactone ring, which is essential for its antitumour activity, and for avoiding premature loss of drug on the way to target camptothecin to the brain. A multivariate approach was used to assess the influence of the qualitative and quantitative composition on the physicochemical properties of camptothecin-loaded SLN in comparison to plain SLN. Mean particle sizes of ≤200 nm, homogenous size distributions and high encapsulation efficiencies (>90%) were achieved for the most suitable formulations. In vitro release studies in plasma, showed a prolonged release profile of camptothecin from SLN, confirming the physical stability of the particles under physiological pH. A higher affinity of the SLN to the porcine brain capillary endothelial cells (BCEC) was shown in comparison to macrophages. MTT studies in BCEC revealed a moderate decrease in the cell viability of camptothecin, when incorporated in SLN compared to free camptothecin in solution. In vivo studies in rats showed that fluorescently labelled SLN were detected in the brain after i.v. administration. This study indicates that the camptothecin-loaded SLN are a promising drug brain delivery system worth to explore further for brain tumour therapy.

Long-term stability, biocompatibility and oral delivery potential of risperidone-loaded solid lipid nanoparticles

A solid lipid nanoparticles (SLN) formulation to improve the oral delivery of risperidone (RISP), a poorly water-soluble drug, was designed and tested. Initially, lipid-RISP solubility was screened to select the best lipid for SLN preparation. Compritol(®)-based formulations were chosen and their long-term stability was assessed over two years of storage (at 25 °C and 4 °C) by means of particle size, polydispersity index (PI), zeta potential (ZP) and encapsulation efficiency (EE) measurements. SLN shape was observed by transmission electron microscopy (TEM) at the beginning and end of the study. The oxidative potential (OP) of the SLN was measured and their biocompatibility with Caco-2 cells was evaluated using the (4,5-dimethylthiazol-2-yl)2,5-dyphenyl-tetrazolium bromide (MTT) assay. In vitro drug release and transport studies were performed to predict the in vivo release profile and to evaluate the drug delivery potential of the SLN formulations, respectively. The RISP-loaded SLN systems were stable and had high EE and similar shape to the placebo formulations before and after storage. Classical Fickian diffusion was identified as the release mechanism for RISP from the SLN formulation. Biocompatibility and dose-dependent RISP transport across Caco-2 cells were observed for the prepared SLN formulations. The viability of SLN as formulations for oral delivery of poorly water-soluble drugs such as RISP was illustrated.

Cell-based in vitro models for predicting drug permeability

Introduction: In vitro cell models have been used to predict drug permeation in early stages of drug development, since they represent an easy and reproducible method, allowing the tracking of drug absorption rate and mechanism, with an advantageous cost–benefit ratio. Such cell-based models are mainly composed of immortalized cells with an intrinsic ability to grow in a monolayer when seeded in permeable supports, maintaining their physiologic characteristics regarding epithelium cell physiology and functionality. Areas covered: This review summarizes the most important intestinal, pulmonary, nasal, vaginal, rectal, ocular and skin cell-based in vitro models for predicting the permeability of drugs. Moreover, the similitude between in vitro cell models and in vivo conditions are discussed, providing evidence that each model may provisionally resemble different drug absorption route. Expert opinion: Despite the widespread use of in vitro cell models for drug permeability and absorption evaluation purposes, a detailed study on the properties of these models and their in vitro–in vivo correlation compared with human data are required to further use in order to consider a future drug discovery optimization and clinical development.

Nanocarriers for pulmonary administration of peptides and therapeutic proteins

Peptides and therapeutic proteins have been the target of intense research and development in recent years by the pharmaceutical and biotechnology industry. Preferably, they are administered through the parenteral route, which is associated with reduced patient compliance. Formulations for noninvasive administration of peptides and therapeutic proteins are currently being developed. Among them, inhalation appears as a promising alternative for the administration of such products. Several formulations for pulmonary delivery are in various stages of development. Despite positive results, conventional formulations have some limitations such as reduced bioavailability and side effects. Nanocarriers may be an alternative way to overcome the problems of conventional formulations. Some nanocarrier-based formulations of peptides and therapeutic proteins are currently under development. The results obtained are promising, revealing the usefulness of these systems in the delivery of such drugs.