João J. Sousa

The size of solid lipid nanoparticles: an interpretation from experimental design.

This study aimed to investigate the role of different factors affecting the size of solid lipid nanoparticles (SLN), prepared by the emulsification-solvent evaporation method. A double factorial design was conducted so as to cover a wide range of sizes, highlighting zones with different behaviour with respect to changes in the controlled variables: lipid concentration, solvent:lipid ratio and emulsifier concentration. The solvent:lipid ratio constituted the main factor influencing particle size. Increasing the amount of solvent induced a decrease in the size. This was a general trend, essentially independent from solvent and lipid type. The amount of emulsifier had a non-trivial impact on size, depending on whether systems were located below, above or close to the optimal surface coverage. The amount of lipid had a limited influence upon particle size, being more relevant for lower lipid concentrations. An optimal formulation was selected for intermediate levels of the three variables. Sonication reduced both particle size and polydispersity. These particles were also tested as drug carriers using simvastatin as a model of lipophilic drug. SLN were able to entrap a high amount of simvastatin, with little effect upon size and zeta potential, constituting a promising carrier for lipophilic drugs.

Co-encapsulating nanostructured lipid carriers for transdermal application: From experimental design to the molecular detail

Co-encapsulation of drugs directed at commonly associated diseases provides a convenient means for administration, especially if transdermally delivered. In this work, a comprehensive study for the co-encapsulation of drugs with a differential lipophilicity, olanzapine and simvastatin, and their transdermal delivery in a formulation containing nanostructured lipid carriers (NLC) is presented. Focus is given to the evaluation of a strategy in which NLC and chemical permeation enhancers are combined. It comprises in vitro, in silico and cellular viability approaches. The optimization and rationalization of the systems are carried out using a two-step factorial design. It is shown that the external medium in the NLC dispersion strongly influences permeation. It is also seen that the use of NLC determines a synergistic effect with selected permeation enhancers, thus promoting marked flux enhancement ratios (48 and 21, respectively for olanzapine and simvastatin) relative to the drugs in solution. The developed formulations can be considered non-irritant. A correlation between enhancer positioning in a lipid bilayer, partially governed by a H-bonding phenomenon, and enhancement effect is suggested from molecular dynamics studies and experimental observations.

The Influence of Drug Physical State on the Dissolution Enhancement of Solid Dispersions Prepared Via Hot-Melt Extrusion: A Case Study Using Olanzapine

In this study, we examine the relationship between the physical structure and dissolution behavior of olanzapine (OLZ) prepared via hot-melt extrusion in three polymers [polyvinylpyrrolidone (PVP) K30, polyvinylpyrrolidone-co-vinyl acetate (PVPVA) 6:4, and Soluplus® (SLP)]. In particular, we examine whether full amorphicity is necessary to achieve a favorable dissolution profile. Drug–polymer miscibility was estimated using melting point depression and Hansen solubility parameters. Solid dispersions were characterized using differential scanning calorimetry, X-ray powder diffraction, and scanning electron microscopy. All the polymers were found to be miscible with OLZ in a decreasing order of PVP>PVPVA>SLP. At a lower extrusion temperature (160°C), PVP generated fully amorphous dispersions with OLZ, whereas the formulations with PVPVA and SLP contained 14%–16% crystalline OLZ. Increasing the extrusion temperature to 180°C allowed the preparation of fully amorphous systems with PVPVA and SLP. Despite these differences, the dissolution rates of these preparations were comparable, with PVP showing a lower release rate despite being fully amorphous. These findings suggested that, at least in the particular case of OLZ, the absence of crystalline material may not be critical to the dissolution performance. We suggest alternative key factors determining dissolution, particularly the dissolution behavior of the polymers themselves.

Identification and Characterization of Stoichiometric and Nonstoichiometric Hydrate Forms of Paroxetine HCl: Reversible Changes in Crystal Dimensions as a Function of Water Absorption

Paroxetine hydrochloride (HCl) is an antidepressant drug, reported to exist in the anhydrous form (form II) and as a stable hemihydrate (form I). In this study, we investigate the hydration behavior of paroxetine HCl form II with a view to understanding both the nature of the interaction with water and the interchange between forms II and I as a function of both temperature and water content. In particular, we present new evidence for both the structure and the interconversion process to be more complex than previously recognized. A combination of characterization techniques was used, including thermal (differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA)), spectroscopic (attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR)), dynamic vapor sorption (DVS) and X-ray powder diffraction (XRPD) with variable humidity, along with computational molecular modeling of the crystal structures. The total amount of water present in form II was surprisingly high (3.8% w/w, 0.8 mol of water/mol of drug), with conversion to the hemihydrate noted on heating in hermetically sealed DSC pans. XRPD, supported by ATR-FTIR and DVS, indicated changes in the unit cell dimensions as a function of water content, with clear evidence for reversible expansion and contraction as a function of relative humidity (RH). Based on these data, we suggest that paroxetine HCl form II is not an anhydrate but rather a nonstoichiometric hydrate. However, no continuous channels are present and, according to molecular modeling simulation, the water is moderately strongly bonded to the crystal, which is in itself an uncommon feature when referring to nonstoichiometric hydrates. Overall, therefore, we suggest that the anhydrous form of paroxetine HCl is not only a nonstoichiometric hydrate but also one that shows highly unusual characteristics in terms of gradual unit cell expansion and contraction despite the absence of continuous channels. These structural features in turn influence the tendency of this drug to convert to the more stable hemihydrate. The study has implications for the recognition and understanding of the behavior of pharmaceutical nonstoichiometric hydrates.

DEVELOPMENT AND VALIDATION OF AN HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF CIS- AND TRANS- PERMETHRIN AND PIPERONYL BUTOXIDE IN PHARMACEUTICAL DOSAGE FORMS

A Reversed-Phase High Performance Liquid Chromatographic (RP-HPLC) method for simultaneous and separate determination of cis and trans-permethrin and piperonyl butoxide in a shampoo formulation is described and fully validated. This method entails the quantification of both components by external standard and ultraviolet detection at two wavelengths (201 and 287nm). A C8 Lichrosorb column and a three component mobile phase, isocratically delivered was used. Results for process validation parameters – selectivity, precision, linearity, and recovery - were in agreement with international specifications. This is a simple method that requires no sample clean up prior to its injection in the system.

Polyphenols from Cymbopogon citratus leaves as topical anti-inflammatory agents.

ETHNOPHARMACOLOGICAL RELEVANCE A variety of plant polyphenols have been reported to have anti-inflammatory, frequently associated with erythema, edema, hyperplasia, skin photoaging and photocarcinogenesis. Cymbopogon citratus (DC). Stapf (Poaceae) is a worldwide known medicinal plant, used in traditional medicine in inflammation-related conditions. AIM OF THE STUDY In this work, the anti-inflammatory potential of C. citratus infusion (CcI) and its polyphenols as topical agents was evaluated in vivo. MATERIALS AND METHODS The plant extract was prepared and its fractioning led two polyphenol-rich fractions: flavonoids fraction (CcF) and tannins fraction (CcT). An oil/water emulsion was developed with each active (CcI, CcF+CcT and diclofenac), pH and texture having been evaluated. Release tests were further performed using static Franz diffusion cells and all collected samples were monitored by HPLC-PDA. In vivo topical anti-inflammatory activity evaluation was performed by the carrageenan-induced rat paw edema model. RESULTS The texture analysis revealed statistically significant differences for all tested parameters to CcF+CcT, supporting its topical application. Release experiments lead to the detection of the phenolic compounds from each sample in the receptor medium and the six major flavonoids were quantified, by HPLC-PDA: carlinoside, isoorientin, cynaroside, luteolin 7-O-neohesperidoside, kurilesin A and cassiaoccidentalin B. The CcF+CcT formulation prompted to the higher release rate for all these flavonoids. CcI4%, CcI1% and CcF+CcT exhibited an edema reduction of 43.18, 29.55 and 59.09%, respectively. CONCLUSIONS Our findings highlight that CcI, containing luteolin 7-O-neohesperidoside, cassiaoccidentalin B, carlinoside, cynaroside and tannins have a potential anti-inflammatory topical activity, suggesting their promising application in the treatment of skin inflammatory pathologies.

A rapid reversed-phase HPLC method for the simultaneous analysis of olanzapine and simvastatin in dual nanostructured lipid carriers

In the present work, a rapid reversed phase high performance liquid chromatography (RP-HPLC) method was developed for the simultaneous determination of simvastatin, including the lactone prodrug (SV) and the respective active hydroxy acid form, simvastatin acid (SVA) and olanzapine (OL) in a formulation containing co-encapsulating-nanostructured lipid carriers (Combo-NLC). The desired chromatographic separation was achieved on a Phenomenex Luna Phenyl-Hexyl, 5 μm (150 × 3 mm) column at a temperature of 35 °C, under isocratic conditions using UV detection at 230 nm. The optimized mobile phase consisted of a mixture of ammonium acetate aqueous solution (0.02 M), methanol and acetonitrile (30 : 35 : 35, v/v/v) at a flow rate of 0.8 mL min−1. The linear regression analysis for the calibration curves showed a good linear correlation over the concentration range 0.5–100 μg mL−1, with determination coefficients, R2, exceeding 0.9994 for all three compounds, SVA, SV and OL. The method was shown to be specific, without the interference of NLC components, precise at the intra-day and inter-day levels, as reflected by the relative standard deviation values, lower than 9.014%, accurate with bias not exceeding 15% and characterized by a recovery rate of 100 ± 8%. The limits of detection and quantification were, respectively, 0.07 and 0.22 μg mL−1 for OL, 0.12 and 0.36 μg mL−1 for SV and 0.09 and 0.27 μg mL−1 for SVA. The method was successfully applied for the determination of Combo-NLC entrapment efficiency.

Generation of hydrate forms of paroxetine HCl from the amorphous state: an evaluation of thermodynamic and experimental predictive approaches.

In this study, we evaluate the use of theoretical thermodynamic analysis of amorphous paroxetine hydrochloride (HCl) as well as experimental assessment in order to identify the most promising approach to stability and dissolution behaviour prediction, particularly in relation to stoichiometric and nonstoichiometric hydrate formation. Differential scanning calorimetry, thermogravimetric analysis, Fourier transform infrared and X-ray diffraction techniques were used. Parameters including heat capacity, configurational thermodynamic quantities, fragility and relaxation time classified amorphous paroxetine HCl as a moderate fragile glass with a considerable degree of molecular mobility. Solubility studies indicated little advantage of the amorphous form over the crystalline due to conversion to the hydrate Form I during equilibration, while the dissolution rate was higher for the amorphous form under sink conditions. A marked difference in the physical stability of amorphous paroxetine HCl was observed between dry and low humidity storage, with the system recrystallizing to the hydrate form. We conclude that, in this particular case (amorphous conversion to the hydrate), water may be playing a dual role in both plasticizing the amorphous form and driving the equilibrium towards the hydrate form, hence prediction of recrystallization behaviour from amorphous characteristics may be confounded by the additional process of hydrate generation.

Modeling of ultra-small lipid nanoparticle surface charge for targeting glioblastoma

&NA; Surface modification of ultra‐small nanostructured lipid carriers (usNLC) via introduction of a positive charge is hypothesized to prompt site‐specific drug delivery for glioblastoma multiforme (GBM) treatment. A more effective interaction with negatively charged lipid bilayers, including the blood‐brain barrier (BBB), will facilitate the nanoparticle access to the brain. For this purpose, usNLC with a particle size of 43.82 ± 0.03 nm and a polydispersity index of 0.224 were developed following a Quality by Design approach. Monomeric and gemini surfactants, either with conventional headgroups or serine‐based ones, were tested for the surface modification, and the respective safety and efficacy to target GBM evaluated. A comprehensive in silico‐in vitro approach is also provided based on molecular dynamics simulations and cytotoxicity studies. Overall, monomeric serine‐derived surfactants displayed the best performance, considering altogether particle size, zeta potential, cytotoxic profile and cell uptake. Although conventional surfactants were able to produce usNLC with suitable physicochemical properties and cell uptake, their use is discouraged due to their high cytotoxicity. This study suggests that monomeric serine‐derived surfactants are promising agents for developing nanosystems aiming at brain drug delivery. Graphical abstract Figure. No caption available.

Targeted Theranostic Nanoparticles for Brain Tumor Treatment

The poor prognosis and rapid recurrence of glioblastoma (GB) are associated to its fast-growing process and invasive nature, which make difficult the complete removal of the cancer infiltrated tissues. Additionally, GB heterogeneity within and between patients demands a patient-focused method of treatment. Thus, the implementation of nanotechnology is an attractive approach considering all anatomic issues of GB, since it will potentially improve brain drug distribution, due to the interaction between the blood–brain barrier and nanoparticles (NPs). In recent years, theranostic techniques have also been proposed and regarded as promising. NPs are advantageous for this application, due to their respective size, easy surface modification and versatility to integrate multiple functional components in one system. The design of nanoparticles focused on therapeutic and diagnostic applications has increased exponentially for the treatment of cancer. This dual approach helps to understand the location of the tumor tissue, the biodistribution of nanoparticles, the progress and efficacy of the treatment, and is highly useful for personalized medicine-based therapeutic interventions. To improve theranostic approaches, different active strategies can be used to modulate the surface of the nanotheranostic particle, including surface markers, proteins, drugs or genes, and take advantage of the characteristics of the microenvironment using stimuli responsive triggers. This review focuses on the different strategies to improve the GB treatment, describing some cell surface markers and their ligands, and reports some strategies, and their efficacy, used in the current research.