Cláudia Nunes

NSAIDs Interactions with Membranes: A Biophysical Approach

This work focuses on the interaction of four representative NSAIDs (nimesulide, indomethacin, meloxicam, and piroxicam) with different membrane models (liposomes, monolayers, and supported lipid bilayers), at different pH values, that mimic the pH conditions of normal (pH 7.4) and inflamed cells (pH 5.0). All models are composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) which is a representative phospholipid of most cellular membranes. Several biophysical techniques were employed: Fluorescence steady-state anisotropy to study the effects of NSAIDs in membrane microviscosity and thus to assess the main phase transition of DPPC, surface pressure-area isotherms to evaluate the adsorption and penetration of NSAIDs into the membrane, IRRAS to acquire structural information of DPPC monolayers upon interaction with the drugs, and AFM to study the changes in surface topography of the lipid bilayers caused by the interaction with NSAIDs. The NSAIDs show pronounced interactions with the lipid membranes at both physiological and inflammatory conditions. Liposomes, monolayers, and supported lipid bilayers experiments allow the conclusion that the pH of the medium is an essential parameter when evaluating drug-membrane interactions, because it conditions the structure of the membrane and the ionization state of NSAIDs, thereby influencing the interactions between these drugs and the lipid membranes. The applied models and techniques provided detailed information about different aspects of the drug-membrane interaction offering valuable information to understand the effect of these drugs on their target membrane-associated enzymes and their side effects at the gastrointestinal level.

Brain targeting effect of camptothecin-loaded solid lipid nanoparticles in rat after intravenous administration

This study intended to investigate the ability of solid lipid nanoparticles (SLN) to deliver camptothecin into the brain parenchyma after crossing the blood-brain barrier. For that purpose, camptothecin-loaded SLN with mean size below 200 nm, low polydispersity index (<0.25), negative surface charge (-20 mV), and high camptothecin association efficiency (>94%) were produced. Synchrotron small and wide angle X-ray scattering (SAXS/WAXS) analysis indicates that SLN maintain their physical stability in contact with DMPC membrane, whereas SLN change the lamellar structure of DMPC into a cubic phase, which is associated with efficient release of the incorporated drugs. Cytotoxicity studies against glioma and macrophage human cell lines revealed that camptothecin-loaded SLN induced cell death with the lowest maximal inhibitory concentration (IC50) values, revealing higher antitumour activity of camptothecin-loaded SLN against gliomas. Furthermore, in vivo biodistribution studies of intravenous camptothecin-loaded SLN performed in rats proved the positive role of SLN on the brain targeting since significant higher brain accumulation of camptothecin was observed, compared to non-encapsulated drug. Pharmacokinetic studies further demonstrated lower deposition of camptothecin in peripheral organs, when encapsulated into SLN, with consequent decrease in potential side toxicological effects. These results confirmed the potential of camptothecin-loaded SLN for antitumour brain treatments.

Interaction of nonsteroidal anti-inflammatory drugs with membranes: In vitro assessment and relevance for their biological actions

Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most commonly used drugs in the world due to their anti-inflammatory, analgesic and antipyretic properties. Nevertheless, the consumption of these drugs is still associated with the occurrence of a wide spectrum of adverse effects. Regarding the major role of membranes in cellular events, the hypothesis that the biological actions of NSAIDs may be related to their effect at the membrane level has triggered the in vitro assessment of NSAIDs-membrane interactions. The use of membrane mimetic models, cell cultures, a wide range of experimental techniques and molecular dynamics simulations has been providing significant information about drugs partition and location within membranes and also about their effect on diverse membrane properties. These studies have indeed been providing evidences that the effect of NSAIDs at membrane level may be an additional mechanism of action and toxicity of NSAIDs. In fact, the pharmacokinetic properties of NSAIDs are closely related to the ability of these drugs to interact and overcome biological membranes. Moreover, the therapeutic actions of NSAIDs may also result from the indirect inhibition of cyclooxygenase due to the disturbing effect of NSAIDs on membrane properties. Furthermore, increasing evidences suggest that the disordering effects of these drugs on membranes may be in the basis of the NSAIDs-induced toxicity in diverse organ systems. Overall, the study of NSAIDs-membrane interactions has proved to be not only important for the better understanding of their pharmacological actions, but also for the rational development of new approaches to overcome NSAIDs adverse effects.

Eradication of Helicobacter pylori: Past, present and future

Helicobacter pylori is the major cause of chronic gastritis and peptic ulcers. Since the classification as a group 1 carcinogenic by International Agency for Research on Cancer, the importance of the complete H. pylori eradication has obtained a novel meaning. Hence, several studies have been made in order to deepen the knowledge in therapy strategies. However, the current therapy presents unsatisfactory eradication rates due to the lack of therapeutic compliance, antibiotic resistance, the degradation of antibiotics at gastric pH and their insufficient residence time in the stomach. Novel approaches have been made in order to overcome these limitations. The purpose of this review is to provide an overview about the current therapy and its limitations, while highlighting the possibility of using micro- and nanotechnology to develop gastric drug delivery systems, overcoming these difficulties in the future.

Lipid-drug interaction: biophysical effects of tolmetin on membrane mimetic systems of different dimensionality.

This work focuses on the application of different biophysical techniques to study the interaction of tolmetin with membrane mimetic models of different dimensionality (liposomes, monolayers, and supported lipid bilayers) composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), used as a representative phospholipid of natural membranes. Several biophysical techniques were employed: Fluorescence steady-state anisotropy to study the effects of NSAIDs on membrane microviscosity and thus to assess the main phase transition of DPPC, surface pressure-area isotherms to evaluate the adsorption and/or penetration of NSAIDs into the membrane, IRRAS to acquire structural information of the lipid membrane upon interaction with the drugs, and AFM to study the dynamic change in surface topography of the lipid bilayers caused by interaction with tolmetin. The experiments were performed taking into account the physiological conditions that tolmetin may find in the course of its in vivo therapeutic activity. Therefore, the studies covered the interactions of tolmetin with lipid membranes in both gel and liquid-crystalline phases at two pH conditions: 7.4 (plasma pH) and 5 (inflamed tissue pH). The applied models and techniques provided detailed information about different aspects of the tolmetin-membrane interaction. The studies have shown that tolmetin-membrane interaction is strongly dependent on the degree of drug ionization and of the lipid phase state, which can be related with the therapeutic action and gastro intestinal toxicity of this drug.

High-throughput microplate assay for the determination of drug partition coefficients

Partition coefficients (Kp) of drugs between the phospholipid bilayer and the aqueous phase provide useful information in quantitative structure-activity relationship studies. Hexadecylphosphocholine (HePC) micelles, composed of a zwitterionic hydrophilic surface and a hydrophobic core, mimic the biomembranes and have several advantages over other lipid structures to assess Kp values. Their preparation is easy, fast and avoids the use of toxic organic solvents, and the output has fewer spectroscopic interferences. Here, we describe a high-throughput microplate protocol for assessing the Kp of drugs using HePC micelles as membrane models and derivative spectrophotometry as the detection technique. Moreover, the time-consuming data treatment to assess Kp values is easily performed by a dedicated Excel routine developed here and described in detail. The Kp values of nonsteroidal anti-inflammatory drugs (acemetacin, clonixin, diclofenac and indomethacin) were determined to show the simplicity of the method and to validate this protocol, which provides Kp values (n = 3) of two drugs in ∼2 h.

A new topical formulation for psoriasis: development of methotrexate-loaded nanostructured lipid carriers.

The aim of the present work was to develop and assess the potential of nanostructured lipid carriers (NLCs) loaded with methotrexate as a new approach for topical therapy of psoriasis. Methotrexate-loaded NLCs were prepared via a modified hot homogenization combined with ultrasonication techniques using either polysorbate 60 (P60) or 80 (P80) as surfactants. The produced NLCs were within the nanosized range (274-298 nm) with relatively low polydispersity index (<0.25) and zeta potential values around -40 mV. NLCs demonstrated storage stability at 25°C up to 28 days. The entrapment efficiency of methotrexate in NLC-P60 and -P80 was ∼65%. Cryo-SEM images showed the spherical shape of the empty and methotrexate-loaded NLCs. FT-IR confirmed methotrexate presence within the NLCs. The in vitro release of methotrexate from the NLCs followed a fast release pattern reaching ∼70% in 2h. In vitro skin penetration study demonstrated that methotrexate-loaded NLCs-P60 had higher skin penetration when compared to free methotrexate, suggesting a significant role of drug-nanocarriers on topical administration. Methotrexate-loaded NLC-P60 provided drug fluxes of 0.88 μg/cm(2)/h, higher (P<0.001) than with the free drug (control, 0.59 μg/cm(2)/h). The results indicate the potential of NLCs for the delivery of methotrexate to topical therapy of psoriasis.

S4(13)-PV cell-penetrating peptide induces physical and morphological changes in membrane-mimetic lipid systems and cell membranes: implications for cell internalization.

The present work aims to gain insights into the role of peptide-lipid interactions in the mechanisms of cellular internalization and endosomal escape of the S4(13)-PV cell-penetrating peptide, which has been successfully used in our laboratory as a nucleic acid delivery system. A S4(13)-PV analogue, S4(13)-PVscr, displaying a scrambled amino acid sequence, deficient cell internalization and drug delivery inability, was used in this study for comparative purposes. Differential scanning calorimetry, fluorescence polarization and X-ray diffraction at small and wide angles techniques showed that both peptides interacted with anionic membranes composed of phosphatidylglycerol or a mixture of this lipid with phosphatidylethanolamine, increasing the lipid order, shifting the phase transition to higher temperatures and raising the correlation length between the bilayers. However, S4(13)-PVscr, in contrast to the wild-type peptide, did not promote lipid domain segregation and induced the formation of an inverted hexagonal lipid phase instead of a cubic phase in the lipid systems assayed. Electron microscopy showed that, as opposed to S4(13)-PVscr, the wild-type peptide induced the formation of a non-lamellar organization in membranes of HeLa cells. We concluded that lateral phase separation and destabilization of membrane lamellar structure without compromising membrane integrity are on the basis of the lipid-driven and receptor-independent mechanism of cell entry of S4(13)-PV peptide. Overall, our results can contribute to a better understanding of the role of peptide-lipid interactions in the mechanisms of cell-penetrating peptide membrane translocation, helping in the future design of more efficient cell-penetrating peptide-based drug delivery systems.

Differential Interactions of Rifabutin with Human and Bacterial Membranes: Implication for Its Therapeutic and Toxic Effects

This work focuses on the interaction of rifabutin (RFB), a naphthalenic ansamycin, with membrane models. Since the therapeutic and toxic effects of this class of drugs are strongly influenced by their lipid affinity, we concerned specifically on the ability of this antibiotic to affect the membrane biophysical properties. The extent of the interaction between RFB and membrane phospholipids was quantified by the partition coefficient (K(p)), using membrane model systems that mimic the human (liposomes of 1,2-dimyristoyl-sn-glycero-phosphocholine, DMPC) and the bacterial (liposomes of 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, DMPG) plasma membranes. To predict the drug location in the membranes, fluorescence quenching and lifetime measurements were carried out using the above-mentioned membrane models labeled with fluorescent probes. Steady-state anisotropy measurements were also performed to evaluate the effect of RFB on the microviscosity of the membranes. Overall, the results support that RFB has higher affinity for the bacterial membrane mediated by electrostatic interactions with the phospholipid head groups.

Biophysics in cancer: The relevance of drug-membrane interaction studies.

Lipidomics has been proving that membrane lipids play a crucial role in several cell functions and are involved in several pathologies, including cancer. In fact, beyond a scaffold where proteins and other components are embedded, the cell membrane can also act as a barrier or a target for anticancer drugs. From this point of view, the development of new chemotherapeutic agents should also take into account the role of the membrane in their activity. This Review aims to highlight the importance of anticancer drug-membrane interactions as a powerful strategy to improve cancer therapy. Biophysical techniques emerge, therefore, as essential tools to unveil such interactions.